Plants having enhanced yield-related traits and a method for making the same

ABSTRACT

The present invention relates generally to the field of molecular biology and concerns a method for enhancing various plant yield-related traits by modulating expression in a plant of a nucleic acid encoding a RHL1 (root hairless 1) polypeptide, a TGase (transglutaminase) polypeptide, a TRY-like (tryptichon) polypeptide, or a BZR (brassinazole-resistant) polypeptide. Constructs useful for performing the methods, as well as plants having enhanced various plant yield-related traits thus obtained, are also provided.

RELATED APPLICATIONS

This application is a continuation of patent application Ser. No.13/000,067 filed on Dec. 20, 2010, which is a national stage application(under 35 U.S.C. §371) of PCT/EP2009/057722, filed Jun. 22, 2009, whichclaims benefit of European application 08159089.5, filed Jun. 26, 2008;U.S. Provisional Application 61/075,909, filed Jun. 26, 2008; EuropeanApplication 08159099.4, filed Jun. 26, 2008; European Application08159093.7, filed Jun. 26, 2008; U.S. Provisional Application61/076,178, filed Jun. 27, 2008; U.S. Provisional Application61/076,158, filed Jun. 27, 2008; European Application 08159746.0, filedJul. 4, 2008 and U.S. Provisional Application 61/078,471 filed on Jul.7, 2008. The entire content of each aforementioned application is herebyincorporated by reference in its entirety.

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by reference intothe specification in its entirety. The name of the text file containingthe Sequence Listing is Sequence_Listing_(—)074040_(—)0100_(—)01. Thesize of the text file is 481 KB, and the text file was created on Jun.17, 2014.

The present invention relates generally to the field of molecularbiology and concerns a method for enhancing various plant yield-relatedtraits by modulating expression in a plant of a nucleic acid encoding aRHL1 (Root Hairless 1). The present invention also concerns plantshaving modulated expression of a nucleic acid encoding a RHL1, whichplants have enhanced various plant yield-related relative tocorresponding wild type plants or other control plants. The inventionalso provides constructs useful in the methods of the invention.

The present invention relates generally to the field of molecularbiology and concerns a method for increasing various plant seedyield-related traits by increasing expression in a plant of a nucleicacid sequence encoding a transglutaminase (TGase) polypeptide. Thepresent invention also concerns plants having increased expression of anucleic acid sequence encoding a TGase polypeptide, which plants haveincreased seed yield-related traits relative to control plants. Theinvention additionally relates to nucleic acid sequences, nucleic acidconstructs, vectors and plants containing said nucleic acid sequences.

The present invention relates generally to the field of molecularbiology and concerns a method for improving various plant growthcharacteristics by modulating expression in a plant of a nucleic acidencoding a TRY-like (Tryptichon) polypeptide. The present invention alsoconcerns plants having modulated expression of a nucleic acid encoding aTRY-like polypeptide, which plants have improved growth characteristicsrelative to corresponding wild type plants or other control plants. Theinvention also provides constructs useful in the methods of theinvention.

The present invention relates generally to the field of molecularbiology and concerns a method for increasing seed yield in plants. Morespecifically, the present invention concerns a method for increasingseed yield in plants by modulating expression in a plant of a nucleicacid encoding a BZR (BRASSINAZOLE-RESISTANT) polypeptide. The presentinvention also concerns plants having modulated expression of a nucleicacid encoding a BZR polypeptide, which plants have increased seed yieldrelative to control plants. The invention also provides hitherto unknownBZR-encoding nucleic acids, and constructs comprising the same, usefulin performing the methods of the invention.

The ever-increasing world population and the dwindling supply of arableland available for agriculture fuels research towards increasing theefficiency of agriculture. Conventional means for crop and horticulturalimprovements utilise selective breeding techniques to identify plantshaving desirable characteristics. However, such selective breedingtechniques have several drawbacks, namely that these techniques aretypically labour intensive and result in plants that often containheterogeneous genetic components that may not always result in thedesirable trait being passed on from parent plants. Advances inmolecular biology have allowed mankind to modify the germplasm ofanimals and plants. Genetic engineering of plants entails the isolationand manipulation of genetic material (typically in the form of DNA orRNA) and the subsequent introduction of that genetic material into aplant. Such technology has the capacity to deliver crops or plantshaving various improved economic, agronomic or horticultural traits.

A trait of particular economic interest is increased yield. Yield isnormally defined as the measurable produce of economic value from acrop. This may be defined in terms of quantity and/or quality. Yield isdirectly dependent on several factors, for example, the number and sizeof the organs, plant architecture (for example, the number of branches),seed production, leaf senescence and more. Root development, nutrientuptake, stress tolerance and early vigour may also be important factorsin determining yield. Optimizing the abovementioned factors maytherefore contribute to increasing crop yield.

Seed yield is a particularly important trait, since the seeds of manyplants are important for human and animal nutrition. Crops such as corn,rice, wheat, canola and soybean account for over half the total humancaloric intake, whether through direct consumption of the seedsthemselves or through consumption of meat products raised on processedseeds. They are also a source of sugars, oils and many kinds ofmetabolites used in industrial processes. Seeds contain an embryo (thesource of new shoots and roots) and an endosperm (the source ofnutrients for embryo growth during germination and during early growthof seedlings). The development of a seed involves many genes, andrequires the transfer of metabolites from the roots, leaves and stemsinto the growing seed. The endosperm, in particular, assimilates themetabolic precursors of carbohydrates, oils and proteins and synthesizesthem into storage macromolecules to fill out the grain.

Plant biomass is yield for forage crops like alfalfa, silage corn andhay. Many proxies for yield have been used in grain crops. Chief amongstthese are estimates of plant size. Plant size can be measured in manyways depending on species and developmental stage, but include totalplant dry weight, above-ground dry weight, above-ground fresh weight,leaf area, stem volume, plant height, rosette diameter, leaf length,root length, root mass, tiller number and leaf number. Many speciesmaintain a conservative ratio between the size of different parts of theplant at a given developmental stage. These allometric relationships areused to extrapolate from one of these measures of size to another (e.g.Tittonell et al 2005 Agric Ecosys & Environ 105: 213). Plant size at anearly developmental stage will typically correlate with plant size laterin development. A larger plant with a greater leaf area can typicallyabsorb more light and carbon dioxide than a smaller plant and thereforewill likely gain a greater weight during the same period (Fasoula &Tollenaar 2005 Maydica 50:39). This is in addition to the potentialcontinuation of the micro-environmental or genetic advantage that theplant had to achieve the larger size initially. There is a stronggenetic component to plant size and growth rate (e.g. ter Steege et al2005 Plant Physiology 139:1078), and so for a range of diverse genotypesplant size under one environmental condition is likely to correlate withsize under another (Hittalmani et al 2003 Theoretical Applied Genetics107:679). In this way a standard environment is used as a proxy for thediverse and dynamic environments encountered at different locations andtimes by crops in the field.

Another important trait for many crops is early vigour. Improving earlyvigour is an important objective of modern rice breeding programs inboth temperate and tropical rice cultivars. Long roots are important forproper soil anchorage in water-seeded rice. Where rice is sown directlyinto flooded fields, and where plants must emerge rapidly through water,longer shoots are associated with vigour. Where drill-seeding ispracticed, longer mesocotyls and coleoptiles are important for goodseedling emergence. The ability to engineer early vigour into plantswould be of great importance in agriculture. For example, poor earlyvigour has been a limitation to the introduction of maize (Zea mays L.)hybrids based on Corn Belt germplasm in the European Atlantic.

Harvest index, the ratio of seed yield to aboveground dry weight, isrelatively stable under many environmental conditions and so a robustcorrelation between plant size and grain yield can often be obtained(e.g. Rebetzke et al 2002 Crop Science 42:739). These processes areintrinsically linked because the majority of grain biomass is dependenton current or stored photosynthetic productivity by the leaves and stemof the plant (Gardener et al 1985 Physiology of Crop Plants. Iowa StateUniversity Press, pp 68-73). Therefore, selecting for plant size, evenat early stages of development, has been used as an indicator for futurepotential yield (e.g. Tittonell et al 2005 Agric Ecosys & Environ 105:213). When testing for the impact of genetic differences on stresstolerance, the ability to standardize soil properties, temperature,water and nutrient availability and light intensity is an intrinsicadvantage of greenhouse or plant growth chamber environments compared tothe field. However, artificial limitations on yield due to poorpollination due to the absence of wind or insects, or insufficient spacefor mature root or canopy growth, can restrict the use of thesecontrolled environments for testing yield differences. Therefore,measurements of plant size in early development, under standardizedconditions in a growth chamber or greenhouse, are standard practices toprovide indication of potential genetic yield advantages.

A further important trait is that of improved abiotic stress tolerance.Abiotic stress is a primary cause of crop loss worldwide, reducingaverage yields for most major crop plants by more than 50% (Wang et al.,Planta (2003) 218: 1-14). Abiotic stresses may be caused by drought,salinity, extremes of temperature, chemical toxicity and oxidativestress. The ability to improve plant tolerance to abiotic stress wouldbe of great economic advantage to farmers worldwide and would allow forthe cultivation of crops during adverse conditions and in territorieswhere cultivation of crops may not otherwise be possible.

Crop yield may therefore be increased by optimising one of theabove-mentioned factors.

Depending on the end use, the modification of certain yield traits maybe favoured over others. For example for applications such as forage orwood production, or bio-fuel resource, an increase in the vegetativeparts of a plant may be desirable, and for applications such as flour,starch or oil production, an increase in seed parameters may beparticularly desirable. Even amongst the seed parameters, some may befavoured over others, depending on the application. Various mechanismsmay contribute to increasing seed yield, whether that is in the form ofincreased seed size or increased seed number.

One approach to increasing yield (seed yield and/or biomass) in plantsmay be through modification of the inherent growth mechanisms of aplant, such as the cell cycle or various signalling pathways involved inplant growth or in defense mechanisms.

Concerning BZR, depending on the end use, the modification of certainyield traits may be favoured over others. For example for applicationssuch as forage or wood production, or bio-fuel resource, an increase inthe vegetative parts of a plant may be desirable, and for applicationssuch as flour, starch or oil production, an increase in seed parametersmay be particularly desirable. Even amongst the seed parameters, somemay be favoured over others, depending on the application. Variousmechanisms may contribute to increasing seed yield, whether that is inthe form of increased seed size or increased seed number or increasenumber of inflorescences.

It has now been found that various plant yield-related may be improvedin plants by modulating expression in a plant of a nucleic acid encodinga RHL1 (Root Hairless 1) in a plant.

Furthermore, it has now been found that various seed yield-relatedtraits may be increased in plants relative to control plants, byincreasing expression in a plant of a nucleic acid sequence encoding atransglutaminase (TGase) polypeptide. The increased seed yield-relatedtraits comprise one or more of: increased total seed yield per plant,increased number of filled seeds, and increased harvest index.

Even furthermore, it has now been found that various growthcharacteristics may be improved in plants by modulating expression in aplant of a nucleic acid encoding a TRY-like (Tryptichon) in a plant.

Yet furthermore, It has now been found that seed yield may be improvedin plants by modulating expression in a plant of a nucleic acid encodinga BZR (BRASSINAZOLE-RESISTANT) polypeptide in a plant.

BACKGROUND 1. Root Hairless 1 (RHL1)

An RHL1 polypeptide was first described in 1998 by Schneider at al.(Genes Dev. 12, 2013-2021) as a nuclear targeted protein required forroot hair initiation in Arabidopsis thaliana. RHL1 polypeptides areubiquitous to the viridiplantae kingdom. Sequence comparison of RHL1originating from different organism reveals that RHL1 polypeptides sharean overall sequence similarity around 30-80% identity. RHL1 polypeptidescomprise a number of putative nuclear localization signals as well asphosphorylation sites and a PEST sequence which is a putativeproteasome-dependent proteins degradation motif. The presence of suchmotifs may reportedly confer some regulatory roles by modulatingsubcellular localization of topos and/or their interaction with otherproteins. The C-terminus of RHL1 proteins has weak but significantsequence similarity to the C-terminal of mammalian Topo II-alpha protein(Sugimoto-Shirasu et al. 2005 PNAS 102, 18736-17741). Eukaryotic topo IIproteins belong to the subclass of the type II topo (typeIIA) that isrequired to unwind replicating double-stranded DNA. Physical Interactionbetween an RHL1 polypeptide and a plant topo VI protein, At TOP6B, hasbeen reported (Sugimoto-Shirasu et al. 2005). It has been suggested thatRHL1 polypeptides function in a plant topo VI complex active during themitotic cell cycle and endocycle of plant cells. Arabidopsis thalianaplants, hyp7, carrying mutations in an RHL1 gene exhibit an extremedwarf phenotype and defects in endoreduplication (Sugimoto-Shirasu etal. 2005).

2. Transglutaminases (TGases)

Transglutaminases (TGases, EC 2.3.2.13;protein-glutamine-gamma-glutamyltransferase) are a family of enzymesthat have a range of calcium (Ca)-dependent catalytic activities, mostof which concern the post-translational modification of proteins. Theycatalyze the covalent attachment to proteins and polypeptides of aseries of substances containing primary amine groups, i.e., they promotethe formation of amide linkages, generally in a Ca-dependent fashion,between the primary amine of an amine donor substrate and they-carboxamide group of peptide-bound

endo-glutamine residues in proteins or polypeptides that are the amineacceptors:

protein glutamine+alkylamine=protein N5−alkylglutamine+NH3.

Polyamines have been shown to serve as physiological substrates ofTGases. Polyamines appear to play an essential role in growth and celldivision process in animals, microorganisms, and plants. One of theroles of polyamines is their regulatory action by a TGase-mediatedprocess of post-translational modification (addition of polyaminemoieties) of enzymes and structural proteins.

TGases enzymes are found intracellularly and extracellularly, and arewidely distributed in bacteria, animals and plants. In plants, the TGaseactivity is found in chloroplasts. Rubisco and apoproteins of theantenna complex have been shown to be substrates of TGase activity,thereby suggesting a role of these enzymes in photosynthesis relatedprocesses, such as protection protection of photosystem antenna proteins(Villalobos et al. (2004) Gene 336: 93-104).

Transgenic rice plants (Claparols et al. (2004) Transgenic Research 13:195-199) expressing a gene encoding rat prostate calcium-dependenttransglutaminase polypeptide under the control of maize constitutivepromoter accumulated the recombinant enzyme in an inactive form.

International patent application WO 2003/102128 describes a nucleic acidsequence encoding a corn TGase polypeptide, vectors, micro-organisms andplants comprising such nucleic acid sequences, and the use ofpolypeptides with such TGase activity in food manipulation, processingand transformation.

Surprisingly, it has now been found that increasing expression in aplant of a nucleic acid sequence encoding a TGase polypeptide as definedherein, gives plants having increased seed yield-related traits relativeto control plants.

According to one embodiment, there is provided a method for increasingseed yield-related traits in plants relative to control plants,comprising increasing expression in a plant of a nucleic acid sequenceencoding a TGase polypeptide as defined herein. The increased seedyield-related traits comprise one or more of: increased total seed yieldper plant, increased number of filled seeds, and increased harvestindex.

3. Tryptichon (TRY-Like)

The Arabidopsis gene Tryptichon encodes a protein that reportedlynegatively regulates trychome development and positively regulates roothair development. Trichome patterning in Arabidopsis is a model for thegeneration of a spacing pattern from initially equivalent cells.Schellmann et al. (EMBO J. 21, 5036-5046, 2002) show that the Tryptichongene that functions in lateral inhibition encodes a single-repeatMYB-related transcription factor that lacks a recognizable activationdomain. It has high sequence similarity to the root hair patterning geneCaprice. Both genes are expressed in trichomes and act together duringlateral inhibition. They further show that Tryptichon and Caprice actredundantly in the position-dependent cell fate determination in theroot epidermis. Thus, the same lateral inhibition mechanism seems to beinvolved in both de novo patterning and position-dependent celldetermination (Schellmann et al., 2002).

4. Brassinazole Resistant1 (BZR1)

The regulation of gene expression is key to the viability of any cell.Several hundreds of proteins are involved in the regulation of genetranscription. In particular transcription factors play a central roleand act directly on gene promoters. Plant genomes devote approximately7% of their coding sequence to transcription factors (TFs; Rushton etal. 2008 Plant Physiology 147:280-295 (2008).

Plants encode a particular class of transcription factors, the BES orBZR proteins, which modulate gene response to fluctuations in plantsteroid hormones such as brassinosteroids (BRs). BZR transcriptionfactors (BZR TFs) are characterized by the presence of a conserved BZR1repressor domain typically found at the N-terminus of the protein andinvolved in binding to the targeted gene promoter. Plant typicallyencode a small number of BZR TFs. For example the Arabidopsis genomecontains only 6 genes encoding BZR TFs, while tobacco, a plant in whichthis family of TFs is expanded encodes 19 BZR TFs. All TFs comprised aconserved BZR1 repressor domain and are predicted to function in themodulation of BR signalling.

In Arabidopsis thaliana, the cascade of events in BR signalling aretriggered upon binding of BRs to the BRASSINOSTEROIDINSENSITIVE1(BRI1)/BK11 receptor complex at the plasma membrane, causingthe release of BKI1. The subsequent dimerization of BRI1 and BRI1ASSOCIATED RECEPTOR KINASE1 (BAK1) activates a downstream signaltransductionpathway that leads to BRI1 EMS SUPPRESSOR1 (BES1) andBRASSINAZOLE RESISTANT1 (BZR1). The phosphorylation of BES1 and BZR1 bythe kinase BIN2 appears to control their signalling activity by actingon the subcelullar localization and stability of the protein.Dephosphorilated BZR1 accumulates in the nuclei which is the site atwhich the transcriptional function is performed (Wang et al., 2006 CellRes. 16: 427-434). Mechanistically, transcription factors of the BZR1family directly bind to the promoter of the targeted gene and may act toactivate or repress expression.

Methods for modulating the Brassinosteroid response pathway to modify anumber of traits in plants have been disclosed (U.S. Pat. No.6,921,848). The traits as defined in U.S. Pat. No. 6,921,848 comprisedincreased growth and cell elongation in various organs and tissues.However those effects did not result in an increase in the number oforgans such as the number of seeds produced and/or in an increase in theseed yield of the plant.

SUMMARY 1. Root Hairless 1 (RHL1)

Surprisingly, it has now been found that modulating expression of anucleic acid encoding a RHL1 polypeptide gives plants having enhancedyield-related traits relative to control plants.

According one embodiment, there is provided a method for enhancing yieldrelated traits of a plant relative to control plants, comprisingmodulating expression of a nucleic acid encoding a RHL1 polypeptide in aplant. The enhanced yield related traits comprised increased earlyvigour, seed yield, number of seed and harvest index of a plant.

2. Tryptichon (TRY-Like)

Surprisingly, it has now been found that modulating expression of anucleic acid encoding a TRY-like polypeptide gives plants havingenhanced yield-related traits in particular increased emergence vigourand/or increased yield relative to control plants.

According one embodiment, there is provided a method for improving yieldrelated traits of a plant relative to control plants, comprisingmodulating expression of a nucleic acid encoding a TRY-like polypeptidein a plant. The improved yield related traits comprised increased seedyield, including total weight of seeds.

3. Brassinazole Resistant1 (BZR1)

Surprisingly, it has now been found that modulating expression of anucleic acid encoding a BZR polypeptide gives plants having increasedseed yield relative to control plants.

According to one embodiment of the invention there is provided a methodfor increasing plant seed yield relative to control plants, comprisingmodulating expression of a nucleic acid encoding a BZR polypeptide in aplant.

DEFINITIONS Polypeptide(s)/Protein(s)

The terms “polypeptide” and “protein” are used interchangeably hereinand refer to amino acids in a polymeric form of any length, linkedtogether by peptide bonds.

Polynucleotide(s)/Nucleic Acid(s)/Nucleic Acid Sequence(s)/NucleotideSequence(s)

The terms “polynucleotide(s)”, “nucleic acid sequence(s)”, “nucleotidesequence(s)”, “nucleic acid(s)”, “nucleic acid molecule” are usedinterchangeably herein and refer to nucleotides, either ribonucleotidesor deoxyribonucleotides or a combination of both, in a polymericunbranched form of any length.

Control Plant(s)

The choice of suitable control plants is a routine part of anexperimental setup and may include corresponding wild type plants orcorresponding plants without the gene of interest. The control plant istypically of the same plant species or even of the same variety as theplant to be assessed. The control plant may also be a nullizygote of theplant to be assessed. Nullizygotes are individuals missing the transgeneby segregation. A “control plant” as used herein refers not only towhole plants, but also to plant parts, including seeds and seed parts.

Homologue(s)

“Homologues” of a protein encompass peptides, oligopeptides,polypeptides, proteins and enzymes having amino acid substitutions,deletions and/or insertions relative to the unmodified protein inquestion and having similar biological and functional activity as theunmodified protein from which they are derived.

A deletion refers to removal of one or more amino acids from a protein.

An insertion refers to one or more amino acid residues being introducedinto a predetermined site in a protein. Insertions may compriseN-terminal and/or C-terminal fusions as well as intra-sequenceinsertions of single or multiple amino acids. Generally, insertionswithin the amino acid sequence will be smaller than N- or C-terminalfusions, of the order of about 1 to 10 residues. Examples of N- orC-terminal fusion proteins or peptides include the binding domain oractivation domain of a transcriptional activator as used in the yeasttwo-hybrid system, phage coat proteins, (histidine)-6-tag, glutathioneS-transferase-tag, protein A, maltose-binding protein, dihydrofolatereductase, Tag•100 epitope, c-myc epitope, FLAG®-epitope, lacZ, CMP(calmodulin-binding peptide), HA epitope, protein C epitope and VSVepitope.

A substitution refers to replacement of amino acids of the protein withother amino acids having similar properties (such as similarhydrophobicity, hydrophilicity, antigenicity, propensity to form orbreak α-helical structures or β-sheet structures). Amino acidsubstitutions are typically of single residues, but may be clustereddepending upon functional constraints placed upon the polypeptide;insertions will usually be of the order of about 1 to 10 amino acidresidues. The amino acid substitutions are preferably conservative aminoacid substitutions. Conservative substitution tables are well known inthe art (see for example Creighton (1984) Proteins. W.H. Freeman andCompany (Eds) and Table 1 below).

TABLE 1 Examples of conserved amino acid substitutions ResidueConservative Substitutions Ala Ser Arg Lys Asn Gln; His Asp Glu Gln AsnCys Ser Glu Asp Gly Pro His Asn; Gln Ile Leu, Val Leu Ile; Val Lys Arg;Gln Met Leu; Ile Phe Met; Leu; Tyr Ser Thr; Gly Thr Ser; Val Trp Tyr TyrTrp; Phe Val Ile; Leu

Amino acid substitutions, deletions and/or insertions may readily bemade using peptide synthetic techniques well known in the art, such assolid phase peptide synthesis and the like, or by recombinant DNAmanipulation. Methods for the manipulation of DNA sequences to producesubstitution, insertion or deletion variants of a protein are well knownin the art. For example, techniques for making substitution mutations atpredetermined sites in DNA are well known to those skilled in the artand include M13 mutagenesis, T7-Gen in vitro mutagenesis (USB,Cleveland, Ohio), QuickChange Site Directed mutagenesis (Stratagene, SanDiego, Calif.), PCR-mediated site-directed mutagenesis or othersite-directed mutagenesis protocols.

Derivatives

“Derivatives” include peptides, oligopeptides, polypeptides which may,compared to the amino acid sequence of the naturally-occurring form ofthe protein, such as the protein of interest, comprise substitutions ofamino acids with non-naturally occurring amino acid residues, oradditions of non-naturally occurring amino acid residues. “Derivatives”of a protein also encompass peptides, oligopeptides, polypeptides whichcomprise naturally occurring altered (glycosylated, acylated,prenylated, phosphorylated, myristoylated, sulphated etc.) ornon-naturally altered amino acid residues compared to the amino acidsequence of a naturally-occurring form of the polypeptide. A derivativemay also comprise one or more non-amino acid substituents or additionscompared to the amino acid sequence from which it is derived, forexample a reporter molecule or other ligand, covalently ornon-covalently bound to the amino acid sequence, such as a reportermolecule which is bound to facilitate its detection, and non-naturallyoccurring amino acid residues relative to the amino acid sequence of anaturally-occurring protein. Furthermore, “derivatives” also includefusions of the naturally-occurring form of the protein with taggingpeptides such as FLAG, HIS6 or thioredoxin (for a review of taggingpeptides, see Terpe, Appl. Microbiol. Biotechnol. 60, 523-533, 2003).

Ortholoque(s)/Paraloque(s)

Orthologues and paralogues encompass evolutionary concepts used todescribe the ancestral relationships of genes. Paralogues are geneswithin the same species that have originated through duplication of anancestral gene; orthologues are genes from different organisms that haveoriginated through speciation, and are also derived from a commonancestral gene.

Domain

The term “domain” refers to a set of amino acids conserved at specificpositions along an alignment of sequences of evolutionarily relatedproteins. While amino acids at other positions can vary betweenhomologues, amino acids that are highly conserved at specific positionsindicate amino acids that are likely essential in the structure,stability or function of a protein. Identified by their high degree ofconservation in aligned sequences of a family of protein homologues,they can be used as identifiers to determine if any polypeptide inquestion belongs to a previously identified polypeptide family.

Motif/Consensus Sequence/Signature

The term “motif” or “consensus sequence” or “signature” refers to ashort conserved region in the sequence of evolutionarily relatedproteins. Motifs are frequently highly conserved parts of domains, butmay also include only part of the domain, or be located outside ofconserved domain (if all of the amino acids of the motif fall outside ofa defined domain).

Hybridisation

The term “hybridisation” as defined herein is a process whereinsubstantially homologous complementary nucleotide sequences anneal toeach other. The hybridisation process can occur entirely in solution,i.e. both complementary nucleic acids are in solution. The hybridisationprocess can also occur with one of the complementary nucleic acidsimmobilised to a matrix such as magnetic beads, Sepharose beads or anyother resin. The hybridisation process can furthermore occur with one ofthe complementary nucleic acids immobilised to a solid support such as anitro-cellulose or nylon membrane or immobilised by e.g.photolithography to, for example, a siliceous glass support (the latterknown as nucleic acid arrays or microarrays or as nucleic acid chips).In order to allow hybridisation to occur, the nucleic acid molecules aregenerally thermally or chemically denatured to melt a double strand intotwo single strands and/or to remove hairpins or other secondarystructures from single stranded nucleic acids.

The term “stringency” refers to the conditions under which ahybridisation takes place. The stringency of hybridisation is influencedby conditions such as temperature, salt concentration, ionic strengthand hybridisation buffer composition. Generally, low stringencyconditions are selected to be about 30° C. lower than the thermalmelting point (T_(m)) for the specific sequence at a defined ionicstrength and pH. Medium stringency conditions are when the temperatureis 20° C. below T_(m), and high stringency conditions are when thetemperature is 10° C. below T_(m). High stringency hybridisationconditions are typically used for isolating hybridising sequences thathave high sequence similarity to the target nucleic acid sequence.However, nucleic acids may deviate in sequence and still encode asubstantially identical polypeptide, due to the degeneracy of thegenetic code. Therefore medium stringency hybridisation conditions maysometimes be needed to identify such nucleic acid molecules.

The Tm is the temperature under defined ionic strength and pH, at which50% of the target sequence hybridises to a perfectly matched probe. TheT_(m) is dependent upon the solution conditions and the base compositionand length of the probe. For example, longer sequences hybridisespecifically at higher temperatures. The maximum rate of hybridisationis obtained from about 16° C. up to 32° C. below T_(m). The presence ofmonovalent cations in the hybridisation solution reduce theelectrostatic repulsion between the two nucleic acid strands therebypromoting hybrid formation; this effect is visible for sodiumconcentrations of up to 0.4M (for higher concentrations, this effect maybe ignored). Formamide reduces the melting temperature of DNA-DNA andDNA-RNA duplexes with 0.6 to 0.7° C. for each percent formamide, andaddition of 50% formamide allows hybridisation to be performed at 30 to45° C., though the rate of hybridisation will be lowered. Base pairmismatches reduce the hybridisation rate and the thermal stability ofthe duplexes. On average and for large probes, the Tm decreases about 1°C. per % base mismatch. The Tm may be calculated using the followingequations, depending on the types of hybrids:

1) DNA-DNA hybrids (Meinkoth and Wahl, Anal. Biochem., 138: 267-284,1984):

T_(m)=81.5° C.+16.6x log₁₀[Na⁺]^(a)+0.41x%[G/C^(b)]−500x[L^(c)]⁻¹−0.61x% formamide

2) DNA-RNA or RNA-RNA hybrids:

Tm=79.8+18.5(log₁₀[Na⁺]^(a))+0.58(% G/C^(b))+11.8(% G/C^(b))²−820/L^(c)

3) oligo-DNA or oligo-RNA^(d) hybrids:

For <20 nucleotides: T _(m)=2(I _(n))

For 20-35 nucleotides: T _(m)=22+1.46(I _(n))

^(a) or for other monovalent cation, but only accurate in the 0.01-0.4 Mrange.^(b) only accurate for % GC in the 30% to 75% range.^(c) L=length of duplex in base pairs.^(d) oligo, oligonucleotide; I_(n),=effective length of primer=2×(no. ofG/C)+(no. of NT).

Non-specific binding may be controlled using any one of a number ofknown techniques such as, for example, blocking the membrane withprotein containing solutions, additions of heterologous RNA, DNA, andSDS to the hybridisation buffer, and treatment with Rnase. Fornon-homologous probes, a series of hybridizations may be performed byvarying one of (i) progressively lowering the annealing temperature (forexample from 68° C. to 42° C.) or (ii) progressively lowering theformamide concentration (for example from 50% to 0%). The skilledartisan is aware of various parameters which may be altered duringhybridisation and which will either maintain or change the stringencyconditions.

Besides the hybridisation conditions, specificity of hybridisationtypically also depends on the function of post-hybridisation washes. Toremove background resulting from non-specific hybridisation, samples arewashed with dilute salt solutions. Critical factors of such washesinclude the ionic strength and temperature of the final wash solution:the lower the salt concentration and the higher the wash temperature,the higher the stringency of the wash. Wash conditions are typicallyperformed at or below hybridisation stringency. A positive hybridisationgives a signal that is at least twice of that of the background.Generally, suitable stringent conditions for nucleic acid hybridisationassays or gene amplification detection procedures are as set forthabove. More or less stringent conditions may also be selected. Theskilled artisan is aware of various parameters which may be alteredduring washing and which will either maintain or change the stringencyconditions.

For example, typical high stringency hybridisation conditions for DNAhybrids longer than 50 nucleotides encompass hybridisation at 65° C. in1×SSC or at 42° C. in 1×SSC and 50% formamide, followed by washing at65° C. in 0.3×SSC. Examples of medium stringency hybridisationconditions for DNA hybrids longer than 50 nucleotides encompasshybridisation at 50° C. in 4×SSC or at 40° C. in 6×SSC and 50%formamide, followed by washing at 50° C. in 2×SSC. The length of thehybrid is the anticipated length for the hybridising nucleic acid. Whennucleic acids of known sequence are hybridised, the hybrid length may bedetermined by aligning the sequences and identifying the conservedregions described herein. 1×SSC is 0.15M NaCl and 15 mM sodium citrate;the hybridisation solution and wash solutions may additionally include5×Denhardt's reagent, 0.5-1.0% SDS, 100 μg/ml denatured, fragmentedsalmon sperm DNA, 0.5% sodium pyrophosphate.

For the purposes of defining the level of stringency, reference can bemade to Sambrook et al. (2001) Molecular Cloning: a laboratory manual,3^(rd) Edition, Cold Spring Harbor Laboratory Press, CSH, New York or toCurrent Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989and yearly updates).

Splice Variant

The term “splice variant” as used herein encompasses variants of anucleic acid sequence in which selected introns and/or exons have beenexcised, replaced, displaced or added, or in which introns have beenshortened or lengthened. Such variants will be ones in which thebiological activity of the protein is substantially retained; this maybe achieved by selectively retaining functional segments of the protein.Such splice variants may be found in nature or may be manmade. Methodsfor predicting and isolating such splice variants are well known in theart (see for example Foissac and Schiex (2005) BMC Bioinformatics 6:25).

Allelic Variant

Alleles or allelic variants are alternative forms of a given gene,located at the same chromosomal position. Allelic variants encompassSingle Nucleotide Polymorphisms (SNPs), as well as SmallInsertion/Deletion Polymorphisms (INDELs). The size of INDELs is usuallyless than 100 bp. SNPs and INDELs form the largest set of sequencevariants in naturally occurring polymorphic strains of most organisms.

Gene Shuffling/Directed Evolution

Gene shuffling or directed evolution consists of iterations of DNAshuffling followed by appropriate screening and/or selection to generatevariants of nucleic acids or portions thereof encoding proteins having amodified biological activity (Castle et al., (2004) Science 304(5674):1151-4; U.S. Pat. Nos. 5,811,238 and 6,395,547).

Regulatory Element/Control Sequence/Promoter

The terms “regulatory element”, “control sequence” and “promoter” areall used interchangeably herein and are to be taken in a broad contextto refer to regulatory nucleic acid sequences capable of effectingexpression of the sequences to which they are ligated. The term“promoter” typically refers to a nucleic acid control sequence locatedupstream from the transcriptional start of a gene and which is involvedin recognising and binding of RNA polymerase and other proteins, therebydirecting transcription of an operably linked nucleic acid. Encompassedby the aforementioned terms are transcriptional regulatory sequencesderived from a classical eukaryotic genomic gene (including the TATA boxwhich is required for accurate transcription initiation, with or withouta CCAAT box sequence) and additional regulatory elements (i.e. upstreamactivating sequences, enhancers and silencers) which alter geneexpression in response to developmental and/or external stimuli, or in atissue-specific manner. Also included within the term is atranscriptional regulatory sequence of a classical prokaryotic gene, inwhich case it may include a −35 box sequence and/or −10 boxtranscriptional regulatory sequences. The term “regulatory element” alsoencompasses a synthetic fusion molecule or derivative that confers,activates or enhances expression of a nucleic acid molecule in a cell,tissue or organ.

A “plant promoter” comprises regulatory elements, which mediate theexpression of a coding sequence segment in plant cells. Accordingly, aplant promoter need not be of plant origin, but may originate fromviruses or micro-organisms, for example from viruses which attack plantcells. The “plant promoter” can also originate from a plant cell, e.g.from the plant which is transformed with the nucleic acid sequence to beexpressed in the inventive process and described herein. This alsoapplies to other “plant” regulatory signals, such as “plant”terminators. The promoters upstream of the nucleotide sequences usefulin the methods of the present invention can be modified by one or morenucleotide substitution(s), insertion(s) and/or deletion(s) withoutinterfering with the functionality or activity of either the promoters,the open reading frame (ORF) or the 3′-regulatory region such asterminators or other 3′ regulatory regions which are located away fromthe ORF. It is furthermore possible that the activity of the promotersis increased by modification of their sequence, or that they arereplaced completely by more active promoters, even promoters fromheterologous organisms. For expression in plants, the nucleic acidmolecule must, as described above, be linked operably to or comprise asuitable promoter which expresses the gene at the right point in timeand with the required spatial expression pattern.

For the identification of functionally equivalent promoters, thepromoter strength and/or expression pattern of a candidate promoter maybe analysed for example by operably linking the promoter to a reportergene and assaying the expression level and pattern of the reporter genein various tissues of the plant. Suitable well-known reporter genesinclude for example beta-glucuronidase or beta-galactosidase. Thepromoter activity is assayed by measuring the enzymatic activity of thebeta-glucuronidase or beta-galactosidase. The promoter strength and/orexpression pattern may then be compared to that of a reference promoter(such as the one used in the methods of the present invention).Alternatively, promoter strength may be assayed by quantifying mRNAlevels or by comparing mRNA levels of the nucleic acid used in themethods of the present invention, with mRNA levels of housekeeping genessuch as 18S rRNA, using methods known in the art, such as Northernblotting with densitometric analysis of autoradiograms, quantitativereal-time PCR or RT-PCR (Heid et al., 1996 Genome Methods 6: 986-994).Generally by “weak promoter” is intended a promoter that drivesexpression of a coding sequence at a low level. By “low level” isintended at levels of about 1/10,000 transcripts to about 1/100,000transcripts, to about 1/500,0000 transcripts per cell. Conversely, a“strong promoter” drives expression of a coding sequence at high level,or at about 1/10 transcripts to about 1/100 transcripts to about 1/1000transcripts per cell. Generally, by “medium strength promoter” isintended a promoter that drives expression of a coding sequence at alower level than a strong promoter, in particular at a level that is inall instances below that obtained when under the control of a 35S CaMVpromoter.

Operably Linked

The term “operably linked” as used herein refers to a functional linkagebetween the promoter sequence and the gene of interest, such that thepromoter sequence is able to initiate transcription of the gene ofinterest.

Constitutive Promoter

A “constitutive promoter” refers to a promoter that is transcriptionallyactive during most, but not necessarily all, phases of growth anddevelopment and under most environmental conditions, in at least onecell, tissue or organ. Table 2a below gives examples of constitutivepromoters.

TABLE 2a Examples of constitutive promoters Gene Source Reference ActinMcElroy et al, Plant Cell, 2: 163-171, 1990 HMGP WO 2004/070039 CAMV 35SOdell et al, Nature, 313: 810-812, 1985 CaMV 19S Nilsson et al.,Physiol. Plant. 100: 456-462, 1997 GOS2 de Pater et al, Plant JNovember; 2(6): 837-44, 1992, WO 2004/065596 Ubiquitin Christensen etal, Plant Mol. Biol. 18: 675-689, 1992 Rice cyclophilin Buchholz et al,Plant Mol Biol. 25(5): 837-43, 1994 Maize H3 histone Lepetit et al, Mol.Gen. Genet. 231: 276-285, 1992 Alfalfa H3 Wu et al. Plant Mol. Biol. 11:641-649, 1988 histone Actin 2 An et al, Plant J. 10(1); 107-121, 199634S FMV Sanger et al., Plant. Mol. Biol., 14, 1990: 433-443 Rubiscosmall U.S. Pat. No. 4,962,028 subunit OCS Leisner (1988) Proc Natl AcadSci USA 85(5): 2553 SAD1 Jain et al., Crop Science, 39 (6), 1999: 1696SAD2 Jain et al., Crop Science, 39 (6), 1999: 1696 nos Shaw et al.(1984) Nucleic Acids Res. 12(20): 7831-7846 V-ATPase WO 01/14572 Superpromoter WO 95/14098 G-box proteins WO 94/12015

Ubiquitous Promoter

A ubiquitous promoter is active in substantially all tissues or cells ofan organism.

Developmentally-Regulated Promoter

A developmentally-regulated promoter is active during certaindevelopmental stages or in parts of the plant that undergo developmentalchanges.

Inducible Promoter

An inducible promoter has induced or increased transcription initiationin response to a chemical (for a review see Gatz 1997, Annu. Rev. PlantPhysiol. Plant Mol. Biol., 48:89-108), environmental or physicalstimulus, or may be “stress-inducible”, i.e. activated when a plant isexposed to various stress conditions, or a “pathogen-inducible” i.e.activated when a plant is exposed to exposure to various pathogens.

Organ-Specific/Tissue-Specific Promoter

An organ-specific or tissue-specific promoter is one that is capable ofpreferentially initiating transcription in certain organs or tissues,such as the leaves, roots, seed tissue etc. For example, a“root-specific promoter” is a promoter that is transcriptionally activepredominantly in plant roots, substantially to the exclusion of anyother parts of a plant, whilst still allowing for any leaky expressionin these other plant parts. Promoters able to initiate transcription incertain cells only are referred to herein as “cell-specific”.

Examples of root-specific promoters are listed in Table 2b below:

TABLE 2b Examples of root-specific promoters Gene Source Reference RCc3Plant Mol Biol. 1995 January; 27(2): 237-48 Arabidopsis PHT1 Kovama etal., 2005; Mudge et al. (2002, Plant J. 31: 341) Medicago phosphate Xiaoet al., 2006 transporter Arabidopsis Pyk10 Nitz et al. (2001) Plant Sci161(2): 337-346 root-expressible genes Tingey et al., EMBO J. 6: 1,1987. tobacco auxin- Van der Zaal et al., Plant Mol. Biol. 16, induciblegene 983, 1991. β-tubulin Oppenheimer, et al., Gene 63: 87, 1988.tobacco root- Conkling, et al., Plant Physiol. 93: 1203, 1990. specificgenes B. napus G1-3b gene U.S. Pat. No. 5,401,836 SbPRP1 Suzuki et al.,Plant Mol. Biol. 21: 109-119, 1993. LRX1 Baumberger et al. 2001, Genes &Dev. 15: 1128 BTG-26 Brassica US 20050044585 napus LeAMT1 (tomato)Lauter et al. (1996, PNAS 3: 8139) The LeNRT1-1 Lauter et al. (1996,PNAS 3: 8139) (tomato) class I patatin Liu et al., Plant Mol. Biol. 153:386-395, 1991. gene (potato) KDC1 (Daucus Downey et al. (2000, J. Biol.Chem. 275: 39420) carota) TobRB7 gene W Song (1997) PhD Thesis, NorthCarolina State University, Raleigh, NC USA OsRAB5a (rice) Wang et al.2002, Plant Sci. 163: 273 ALF5 (Arabidopsis) Diener et al. (2001, PlantCell 13: 1625) NRT2; 1Np (N. Quesada et al. (1997, Plant Mol. Biol. 34:265) plumbaginifolia)

A seed-specific promoter is transcriptionally active predominantly inseed tissue, but not necessarily exclusively in seed tissue (in cases ofleaky expression). The seed-specific promoter may be active during seeddevelopment and/or during germination. The seed specific promoter may beendosperm/aleurone/embryo specific. Examples of seed-specific promoters(endosperm/aleurone/embryo specific) are shown in Table 2c to Table 2fbelow. Further examples of seed-specific promoters are given in Qing Quand Takaiwa (Plant Biotechnol. J. 2, 113-125, 2004), which disclosure isincorporated by reference herein as if fully set forth.

TABLE 2c Examples of seed-specific promoters Gene source Referenceseed-specific genes Simon et al., Plant Mol. Biol. 5: 191, 1985;Scofield et al., J. Biol. Chem. 262: 12202, 1987.; Baszczynski et al.,Plant Mol. Biol. 14: 633, 1990. Brazil Nut albumin Pearson et al., PlantMol. Biol. 18: 235-245, 1992. legumin Ellis et al., Plant Mol. Biol. 10:203-214, 1988. glutelin (rice) Takaiwa et al., Mol. Gen. Genet. 208:15-22, 1986; Takaiwa et al., FEBS Letts. 221: 43-47, 1987. zein Matzkeet al Plant Mol Biol, 14(3): 323-32 1990 napA Stalberg et al, Planta199: 515-519, 1996. wheat LMW and HMW Mol Gen Genet 216: 81-90, 1989;NAR 17: 461-2, 1989 glutenin-1 wheat SPA Albani et al, Plant Cell, 9:171-184, 1997 wheat α, β, γ-gliadins EMBO J. 3: 1409-15, 1984 barleyItr1 promoter Diaz et al. (1995) Mol Gen Genet 248(5): 592-8 barley B1,C, D, hordein Theor Appl Gen 98: 1253-62, 1999; Plant J 4: 343-55, 1993;Mol Gen Genet 250: 750-60, 1996 barley DOF Mena et al, The PlantJournal, 116(1): 53-62, 1998 blz2 EP99106056.7 synthetic promoterVicente-Carbajosa et al., Plant J. 13: 629-640, 1998. rice prolaminNRP33 Wu et al, Plant Cell Physiology 39(8) 885-889, 1998 ricea-globulin Glb-1 Wu et al, Plant Cell Physiology 39(8) 885-889, 1998rice OSH1 Sato et al, Proc. Natl. Acad. Sci. USA, 93: 8117-8122, 1996rice α-globulin REB/OHP-1 Nakase et al. Plant Mol. Biol. 33: 513-522,1997 rice ADP-glucose pyrophos- Trans Res 6: 157-68, 1997 phorylasemaize ESR gene family Plant J 12: 235-46, 1997 sorghum α-kafirin DeRoseet al., Plant Mol. Biol 32: 1029-35, 1996 KNOX Postma-Haarsma et al,Plant Mol. Biol. 39: 257-71, 1999 rice oleosin Wu et al, J. Biochem.123: 386, 1998 sunflower oleosin Cummins et al., Plant Mol. Biol. 19:873-876, 1992 PRO0117, putative rice 40S WO 2004/070039 ribosomalprotein PRO0136, rice alanine unpublished aminotransferase PRO0147,trypsin inhibitor unpublished ITR1 (barley) PRO0151, rice WSI18 WO2004/070039 PRO0175, rice RAB21 WO 2004/070039 PRO005 WO 2004/070039PRO0095 WO 2004/070039 α-amylase (Amy32b) Lanahan et al, Plant Cell 4:203-211, 1992; Skriver et al, Proc Natl Acad Sci USA 88: 7266-7270, 1991cathepsin β-like gene Cejudo et al, Plant Mol Biol 20: 849-856, 1992Barley Ltp2 Kalla et al., Plant J. 6: 849-60, 1994 Chi26 Leah et al.,Plant J. 4: 579-89, 1994 Maize B-Peru Selinger et al., Genetics 149;1125-38, 1998

TABLE 2d examples of endosperm-specific promoters Gene source Referenceglutelin (rice) Takaiwa et al. (1986) Mol Gen Genet 208: 15-22; Takaiwaet al. (1987) FEBS Letts. 221: 43-47 zein Matzke et al., (1990) PlantMol Biol 14(3): 323-32 wheat LMW Colot et al. (1989) Mol Gen Genet 216:81-90, and HMW Anderson et al. (1989) NAR 17: 461-2 glutenin-1 wheat SPAAlbani et al. (1997) Plant Cell 9: 171-184 wheat gliadins Rafalski etal. (1984) EMBO 3: 1409-15 barley Itr1 Diaz et al. (1995) Mol Gen Genet248(5): 592-8 promoter barley B1, C, D, Cho et al. (1999) Theor ApplGenet 98: 1253-62; hordein Muller et al. (1993) Plant J 4: 343-55;Sorenson et al. (1996) Mol Gen Genet 250: 750-60 barley DOF Mena et al,(1998) Plant J 116(1): 53-62 blz2 Onate et al. (1999) J Biol Chem274(14): 9175-82 synthetic promoter Vicente-Carbajosa et al. (1998)Plant J 13: 629-640 rice prolamin Wu et al, (1998) Plant Cell Physiol39(8) 885-889 NRP33 rice globulin Wu et al. (1998) Plant Cell Physiol39(8) 885-889 Glb-1 rice globulin Nakase et al. (1997) Plant Molec Biol33: 513-522 REB/OHP-1 rice ADP-glucose Russell et al. (1997) Trans Res6: 157-68 pyrophosphorylase maize ESR Opsahl-Ferstad et al. (1997) PlantJ 12: 235-46 gene family sorghum kafirin DeRose et al. (1996) Plant MolBiol 32: 1029-35

TABLE 2e Examples of embryo specific promoters: Gene source Referencerice OSH1 Sato et al, Proc. Natl. Acad. Sci. USA, 93: 8117-8122, 1996KNOX Postma-Haarsma et al, Plant Mol. Biol. 39: 257-71, 1999 PRO0151 WO2004/070039 PRO0175 WO 2004/070039 PRO005 WO 2004/070039 PRO0095 WO2004/070039

TABLE 2f Examples of aleurone-specific promoters: Gene source Referenceα-amylase Lanahan et al, Plant Cell 4: 203-211, 1992; (Amy32b) Skriveret al, Proc Natl Acad Sci USA 88: 7266-7270, 1991 cathepsin β-like geneCejudo et al, Plant Mol Biol 20: 849-856, 1992 Barley Ltp2 Kalla et al.,Plant J. 6: 849-60, 1994 Chi26 Leah et al., Plant J. 4: 579-89, 1994Maize B-Peru Selinger et al., Genetics 149; 1125-38, 1998

A green tissue-specific promoter as defined herein is a promoter that istranscriptionally active predominantly in green tissue, substantially tothe exclusion of any other parts of a plant, whilst still allowing forany leaky expression in these other plant parts.

Examples of green tissue-specific promoters which may be used to performthe methods of the invention are shown in Table 2g below.

TABLE 2g Examples of green tissue-specific promoters Gene ExpressionReference Maize Orthophosphate dikinase Leaf specific Fukavama et al.,2001 Maize Phosphoenolpyruvate Leaf specific Kausch et al., 2001carboxylase Rice Phosphoenolpyruvate Leaf specific Liu et al., 2003carboxylase Rice small subunit Rubisco Leaf specific Nomura et al., 2000rice beta expansin EXBP9 Shoot specific WO 2004/070039 Pigeonpea smallsubunit Rubisco Leaf specific Panguluri et al., 2005 Pea RBCS3A Leafspecific

Another example of a tissue-specific promoter is a meristem-specificpromoter, which is transcriptionally active predominantly inmeristematic tissue, substantially to the exclusion of any other partsof a plant, whilst still allowing for any leaky expression in theseother plant parts. Examples of green meristem-specific promoters whichmay be used to perform the methods of the invention are shown in Table2h below.

TABLE 2h Examples of meristem-specific promoters Gene source Expressionpattern Reference rice OSH1 Shoot apical meristem, Sato et al. (1996)from embryo globular Proc. Natl. Acad. Sci. stage to seedling stage USA,93: 8117-8122 Rice metallothionein Meristem specific BAD87835.1 WAK1 &WAK 2 Shoot and root apical Wagner & Kohorn meristems, and in ex- (2001)Plant Cell panding leaves and sepals 13(2): 303-318

Terminator

The term “terminator” encompasses a control sequence which is a DNAsequence at the end of a transcriptional unit which signals 3′processing and polyadenylation of a primary transcript and terminationof transcription. The terminator can be derived from the natural gene,from a variety of other plant genes, or from T-DNA. The terminator to beadded may be derived from, for example, the nopaline synthase oroctopine synthase genes, or alternatively from another plant gene, orless preferably from any other eukaryotic gene.

Modulation

The term “modulation” means in relation to expression or geneexpression, a process in which the expression level is changed by saidgene expression in comparison to the control plant, the expression levelmay be increased or decreased. The original, unmodulated expression maybe of any kind of expression of a structural RNA (rRNA, tRNA) or mRNAwith subsequent translation. The term “modulating the activity” shallmean any change of the expression of the inventive nucleic acidsequences or encoded proteins, which leads to increased yield and/orincreased growth of the plants.

Expression

The term “expression” or “gene expression” means the transcription of aspecific gene or specific genes or specific genetic construct. The term“expression” or “gene expression” in particular means the transcriptionof a gene or genes or genetic construct into structural RNA (rRNA, tRNA)or mRNA with or without subsequent translation of the latter into aprotein. The process includes transcription of DNA and processing of theresulting mRNA product.

Increased Expression/Overexpression

The term “increased expression” or “overexpression” as used herein meansany form of expression that is additional to the original wild-typeexpression level.

Methods for increasing expression of genes or gene products are welldocumented in the art and include, for example, overexpression driven byappropriate promoters, the use of transcription enhancers or translationenhancers. Isolated nucleic acids which serve as promoter or enhancerelements may be introduced in an appropriate position (typicallyupstream) of a non-heterologous form of a polynucleotide so as toupregulate expression of a nucleic acid encoding the polypeptide ofinterest. For example, endogenous promoters may be altered in vivo bymutation, deletion, and/or substitution (see, Kmiec, U.S. Pat. No.5,565,350; Zarling et al., WO9322443), or isolated promoters may beintroduced into a plant cell in the proper orientation and distance froma gene of the present invention so as to control the expression of thegene.

If polypeptide expression is desired, it is generally desirable toinclude a polyadenylation region at the 3′-end of a polynucleotidecoding region. The polyadenylation region can be derived from thenatural gene, from a variety of other plant genes, or from T-DNA. The 3′end sequence to be added may be derived from, for example, the nopalinesynthase or octopine synthase genes, or alternatively from another plantgene, or less preferably from any other eukaryotic gene.

An intron sequence may also be added to the 5′ untranslated region (UTR)or the coding sequence of the partial coding sequence to increase theamount of the mature message that accumulates in the cytosol. Inclusionof a spliceable intron in the transcription unit in both plant andanimal expression constructs has been shown to increase gene expressionat both the mRNA and protein levels up to 1000-fold (Buchman and Berg(1988) Mol. Cell biol. 8: 4395-4405; Callis et al. (1987) Genes Dev1:1183-1200). Such intron enhancement of gene expression is typicallygreatest when placed near the 5′ end of the transcription unit. Use ofthe maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron areknown in the art. For general information see: The Maize Handbook,Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).

Endogenous Gene

Reference herein to an “endogenous” gene not only refers to the gene inquestion as found in a plant in its natural form (i.e., without therebeing any human intervention), but also refers to that same gene (or asubstantially homologous nucleic acid/gene) in an isolated formsubsequently (re)introduced into a plant (a transgene). For example, atransgenic plant containing such a transgene may encounter a substantialreduction of the transgene expression and/or substantial reduction ofexpression of the endogenous gene. The isolated gene may be isolatedfrom an organism or may be manmade, for example by chemical synthesis.

Decreased Expression

Reference herein to “decreased expression” or “reduction or substantialelimination” of expression is taken to mean a decrease in endogenousgene expression and/or polypeptide levels and/or polypeptide activityrelative to control plants. The reduction or substantial elimination isin increasing order of preference at least 10%, 20%, 30%, 40% or 50%,60%, 70%, 80%, 85%, 90%, or 95%, 96%, 97%, 98%, 99% or more reducedcompared to that of control plants. Methods for decreasing expressionare known in the art and the skilled person would readily be able toadapt the known methods for silencing so as to achieve reduction ofexpression of an endogenous gene in a whole plant or in parts thereofthrough the use of an appropriate promoter, for example.

For the reduction or substantial elimination of expression an endogenousgene in a plant, a sufficient length of substantially contiguousnucleotides of a nucleic acid sequence is required. In order to performgene silencing, this may be as little as 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10 or fewer nucleotides, alternatively this may be as much asthe entire gene (including the 5′ and/or 3′ UTR, either in part or inwhole). The stretch of substantially contiguous nucleotides may bederived from the nucleic acid encoding the protein of interest (targetgene), or from any nucleic acid capable of encoding an orthologue,paralogue or homologue of the protein of interest. Preferably, thestretch of substantially contiguous nucleotides is capable of forminghydrogen bonds with the target gene (either sense or antisense strand),more preferably, the stretch of substantially contiguous nucleotideshas, in increasing order of preference, 50%, 60%, 70%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, 100% sequence identity to the target gene(either sense or antisense strand). A nucleic acid sequence encoding a(functional) polypeptide is not a requirement for the various methodsdiscussed herein for the reduction or substantial elimination ofexpression of an endogenous gene.

Examples of various methods for the reduction or substantial eliminationof expression in a plant of an endogenous gene, or for lowering levelsand/or activity of a protein, are known to the skilled in the art. Askilled person would readily be able to adapt the known methods forsilencing, so as to achieve reduction of expression of an endogenousgene in a whole plant or in parts thereof through the use of anappropriate promoter, for example.

This reduction or substantial elimination of expression may be achievedusing routine tools and techniques. A preferred method for the reductionor substantial elimination of endogenous gene expression is byintroducing and expressing in a plant a genetic construct into which thenucleic acid (in this case a stretch of substantially contiguousnucleotides derived from the gene of interest, or from any nucleic acidcapable of encoding an orthologue, paralogue or homologue of any one ofthe protein of interest) is cloned as an inverted repeat (in part orcompletely), separated by a spacer (non-coding DNA).

In such a preferred method, expression of the endogenous gene is reducedor substantially eliminated through RNA-mediated silencing using aninverted repeat of a nucleic acid or a part thereof (in this case astretch of substantially contiguous nucleotides derived from the gene ofinterest, or from any nucleic acid capable of encoding an orthologue,paralogue or homologue of the protein of interest), preferably capableof forming a hairpin structure. The inverted repeat is cloned in anexpression vector comprising control sequences. A non-coding DNA nucleicacid sequence (a spacer, for example a matrix attachment region fragment(MAR), an intron, a polylinker, etc.) is located between the twoinverted nucleic acids forming the inverted repeat. After transcriptionof the inverted repeat, a chimeric RNA with a self-complementarystructure is formed (partial or complete). This double-stranded RNAstructure is referred to as the hairpin RNA (hpRNA). The hpRNA isprocessed by the plant into siRNAs that are incorporated into anRNA-induced silencing complex (RISC). The RISC further cleaves the mRNAtranscripts, thereby substantially reducing the number of mRNAtranscripts to be translated into polypeptides. For further generaldetails see for example, Grierson et al. (1998) WO 98/53083; Waterhouseet al. (1999) WO 99/53050).

Performance of the methods of the invention does not rely on introducingand expressing in a plant a genetic construct into which the nucleicacid is cloned as an inverted repeat, but any one or more of severalwell-known “gene silencing” methods may be used to achieve the sameeffects.

One such method for the reduction of endogenous gene expression isRNA-mediated silencing of gene expression (downregulation). Silencing inthis case is triggered in a plant by a double stranded RNA sequence(dsRNA) that is substantially similar to the target endogenous gene.This dsRNA is further processed by the plant into about 20 to about 26nucleotides called short interfering RNAs (siRNAs). The siRNAs areincorporated into an RNA-induced silencing complex (RISC) that cleavesthe mRNA transcript of the endogenous target gene, thereby substantiallyreducing the number of mRNA transcripts to be translated into apolypeptide. Preferably, the double stranded RNA sequence corresponds toa target gene.

Another example of an RNA silencing method involves the introduction ofnucleic acid sequences or parts thereof (in this case a stretch ofsubstantially contiguous nucleotides derived from the gene of interest,or from any nucleic acid capable of encoding an orthologue, paralogue orhomologue of the protein of interest) in a sense orientation into aplant. “Sense orientation” refers to a DNA sequence that is homologousto an mRNA transcript thereof. Introduced into a plant would thereforebe at least one copy of the nucleic acid sequence. The additionalnucleic acid sequence will reduce expression of the endogenous gene,giving rise to a phenomenon known as co-suppression. The reduction ofgene expression will be more pronounced if several additional copies ofa nucleic acid sequence are introduced into the plant, as there is apositive correlation between high transcript levels and the triggeringof co-suppression.

Another example of an RNA silencing method involves the use of antisensenucleic acid sequences. An “antisense” nucleic acid sequence comprises anucleotide sequence that is complementary to a “sense” nucleic acidsequence encoding a protein, i.e. complementary to the coding strand ofa double-stranded cDNA molecule or complementary to an mRNA transcriptsequence. The antisense nucleic acid sequence is preferablycomplementary to the endogenous gene to be silenced. The complementaritymay be located in the “coding region” and/or in the “non-coding region”of a gene. The term “coding region” refers to a region of the nucleotidesequence comprising codons that are translated into amino acid residues.The term “non-coding region” refers to 5′ and 3′ sequences that flankthe coding region that are transcribed but not translated into aminoacids (also referred to as 5′ and 3′ untranslated regions).

Antisense nucleic acid sequences can be designed according to the rulesof Watson and Crick base pairing. The antisense nucleic acid sequencemay be complementary to the entire nucleic acid sequence (in this case astretch of substantially contiguous nucleotides derived from the gene ofinterest, or from any nucleic acid capable of encoding an orthologue,paralogue or homologue of the protein of interest), but may also be anoligonucleotide that is antisense to only a part of the nucleic acidsequence (including the mRNA 5′ and 3′ UTR). For example, the antisenseoligonucleotide sequence may be complementary to the region surroundingthe translation start site of an mRNA transcript encoding a polypeptide.The length of a suitable antisense oligonucleotide sequence is known inthe art and may start from about 50, 45, 40, 35, 30, 25, 20, 15 or 10nucleotides in length or less. An antisense nucleic acid sequenceaccording to the invention may be constructed using chemical synthesisand enzymatic ligation reactions using methods known in the art. Forexample, an antisense nucleic acid sequence (e.g., an antisenseoligonucleotide sequence) may be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acid sequences, e.g., phosphorothioate derivatives and acridinesubstituted nucleotides may be used. Examples of modified nucleotidesthat may be used to generate the antisense nucleic acid sequences arewell known in the art. Known nucleotide modifications includemethylation, cyclization and ‘caps’ and substitution of one or more ofthe naturally occurring nucleotides with an analogue such as inosine.Other modifications of nucleotides are well known in the art.

The antisense nucleic acid sequence can be produced biologically usingan expression vector into which a nucleic acid sequence has beensubcloned in an antisense orientation (i.e., RNA transcribed from theinserted nucleic acid will be of an antisense orientation to a targetnucleic acid of interest). Preferably, production of antisense nucleicacid sequences in plants occurs by means of a stably integrated nucleicacid construct comprising a promoter, an operably linked antisenseoligonucleotide, and a terminator.

The nucleic acid molecules used for silencing in the methods of theinvention (whether introduced into a plant or generated in situ)hybridize with or bind to mRNA transcripts and/or genomic DNA encoding apolypeptide to thereby inhibit expression of the protein, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid sequence which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. Antisense nucleic acid sequences may be introducedinto a plant by transformation or direct injection at a specific tissuesite. Alternatively, antisense nucleic acid sequences can be modified totarget selected cells and then administered systemically. For example,for systemic administration, antisense nucleic acid sequences can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid sequence to peptides or antibodies which bind to cellsurface receptors or antigens. The antisense nucleic acid sequences canalso be delivered to cells using the vectors described herein.

According to a further aspect, the antisense nucleic acid sequence is ana-anomeric nucleic acid sequence. An a-anomeric nucleic acid sequenceforms specific double-stranded hybrids with complementary RNA in which,contrary to the usual b-units, the strands run parallel to each other(Gaultier et al. (1987) Nucl Ac Res 15: 6625-6641). The antisensenucleic acid sequence may also comprise a 2′-o-methylribonucleotide(Inoue et al. (1987) Nucl Ac Res 15, 6131-6148) or a chimeric RNA-DNAanalogue (Inoue et al. (1987) FEBS Lett. 215, 327-330).

The reduction or substantial elimination of endogenous gene expressionmay also be performed using ribozymes. Ribozymes are catalytic RNAmolecules with ribonuclease activity that are capable of cleaving asingle-stranded nucleic acid sequence, such as an mRNA, to which theyhave a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes(described in Haselhoff and Gerlach (1988) Nature 334, 585-591) can beused to catalytically cleave mRNA transcripts encoding a polypeptide,thereby substantially reducing the number of mRNA transcripts to betranslated into a polypeptide. A ribozyme having specificity for anucleic acid sequence can be designed (see for example: Cech et al. U.S.Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742).Alternatively, mRNA transcripts corresponding to a nucleic acid sequencecan be used to select a catalytic RNA having a specific ribonucleaseactivity from a pool of RNA molecules (Bartel and Szostak (1993) Science261, 1411-1418). The use of ribozymes for gene silencing in plants isknown in the art (e.g., Atkins et al. (1994) WO 94/00012; Lenne et al.(1995) WO 95/03404; Lutziger et al. (2000) WO 00/00619; Prinsen et al.(1997) WO 97/13865 and Scott et al. (1997) WO 97/38116).

Gene silencing may also be achieved by insertion mutagenesis (forexample, T-DNA insertion or transposon insertion) or by strategies asdescribed by, among others, Angell and Baulcombe ((1999) Plant J 20(3):357-62), (Amplicon VIGS WO 98/36083), or Baulcombe (WO 99/15682).

Gene silencing may also occur if there is a mutation on an endogenousgene and/or a mutation on an isolated gene/nucleic acid subsequentlyintroduced into a plant. The reduction or substantial elimination may becaused by a non-functional polypeptide. For example, the polypeptide maybind to various interacting proteins; one or more mutation(s) and/ortruncation(s) may therefore provide for a polypeptide that is still ableto bind interacting proteins (such as receptor proteins) but that cannotexhibit its normal function (such as signalling ligand).

A further approach to gene silencing is by targeting nucleic acidsequences complementary to the regulatory region of the gene (e.g., thepromoter and/or enhancers) to form triple helical structures thatprevent transcription of the gene in target cells. See Helene, C.,Anticancer Drug Res. 6, 569-84, 1991; Helene et al., Ann. N.Y. Acad.Sci. 660, 27-36 1992; and Maher, L. J. Bioassays 14, 807-15, 1992.

Other methods, such as the use of antibodies directed to an endogenouspolypeptide for inhibiting its function in planta, or interference inthe signalling pathway in which a polypeptide is involved, will be wellknown to the skilled man. In particular, it can be envisaged thatmanmade molecules may be useful for inhibiting the biological functionof a target polypeptide, or for interfering with the signalling pathwayin which the target polypeptide is involved.

Alternatively, a screening program may be set up to identify in a plantpopulation natural variants of a gene, which variants encodepolypeptides with reduced activity. Such natural variants may also beused for example, to perform homologous recombination.

Artificial and/or natural microRNAs (miRNAs) may be used to knock outgene expression and/or mRNA translation. Endogenous miRNAs are singlestranded small RNAs of typically 19-24 nucleotides long. They functionprimarily to regulate gene expression and/or mRNA translation. Mostplant microRNAs (miRNAs) have perfect or near-perfect complementaritywith their target sequences. However, there are natural targets with upto five mismatches. They are processed from longer non-coding RNAs withcharacteristic fold-back structures by double-strand specific RNases ofthe Dicer family. Upon processing, they are incorporated in theRNA-induced silencing complex (RISC) by binding to its main component,an Argonaute protein. MiRNAs serve as the specificity components ofRISC, since they base-pair to target nucleic acids, mostly mRNAs, in thecytoplasm. Subsequent regulatory events include target mRNA cleavage anddestruction and/or translational inhibition. Effects of miRNAoverexpression are thus often reflected in decreased mRNA levels oftarget genes.

Artificial microRNAs (amiRNAs), which are typically 21 nucleotides inlength, can be genetically engineered specifically to negativelyregulate gene expression of single or multiple genes of interest.Determinants of plant microRNA target selection are well known in theart. Empirical parameters for target recognition have been defined andcan be used to aid in the design of specific amiRNAs, (Schwab et al.,Dev. Cell 8, 517-527, 2005). Convenient tools for design and generationof amiRNAs and their precursors are also available to the public (Schwabet al., Plant Cell 18, 1121-1133, 2006).

For optimal performance, the gene silencing techniques used for reducingexpression in a plant of an endogenous gene requires the use of nucleicacid sequences from monocotyledonous plants for transformation ofmonocotyledonous plants, and from dicotyledonous plants fortransformation of dicotyledonous plants. Preferably, a nucleic acidsequence from any given plant species is introduced into that samespecies. For example, a nucleic acid sequence from rice is transformedinto a rice plant. However, it is not an absolute requirement that thenucleic acid sequence to be introduced originates from the same plantspecies as the plant in which it will be introduced. It is sufficientthat there is substantial homology between the endogenous target geneand the nucleic acid to be introduced.

Described above are examples of various methods for the reduction orsubstantial elimination of expression in a plant of an endogenous gene.A person skilled in the art would readily be able to adapt theaforementioned methods for silencing so as to achieve reduction ofexpression of an endogenous gene in a whole plant or in parts thereofthrough the use of an appropriate promoter, for example.

Selectable Marker (Gene)/Reporter Gene

“Selectable marker”, “selectable marker gene” or “reporter gene”includes any gene that confers a phenotype on a cell in which it isexpressed to facilitate the identification and/or selection of cellsthat are transfected or transformed with a nucleic acid construct of theinvention. These marker genes enable the identification of a successfultransfer of the nucleic acid molecules via a series of differentprinciples. Suitable markers may be selected from markers that conferantibiotic or herbicide resistance, that introduce a new metabolic traitor that allow visual selection. Examples of selectable marker genesinclude genes conferring resistance to antibiotics (such as nptII thatphosphorylates neomycin and kanamycin, or hpt, phosphorylatinghygromycin, or genes conferring resistance to, for example, bleomycin,streptomycin, tetracyclin, chloramphenicol, ampicillin, gentamycin,geneticin (G418), spectinomycin or blasticidin), to herbicides (forexample bar which provides resistance to Basta®; aroA or gox providingresistance against glyphosate, or the genes conferring resistance to,for example, imidazolinone, phosphinothricin or sulfonylurea), or genesthat provide a metabolic trait (such as manA that allows plants to usemannose as sole carbon source or xylose isomerase for the utilisation ofxylose, or antinutritive markers such as the resistance to2-deoxyglucose). Expression of visual marker genes results in theformation of colour (for example β-glucuronidase, GUS or β-galactosidasewith its coloured substrates, for example X-Gal), luminescence (such asthe luciferin/luceferase system) or fluorescence (Green FluorescentProtein, GFP, and derivatives thereof). This list represents only asmall number of possible markers. The skilled worker is familiar withsuch markers. Different markers are preferred, depending on the organismand the selection method.

It is known that upon stable or transient integration of nucleic acidsinto plant cells, only a minority of the cells takes up the foreign DNAand, if desired, integrates it into its genome, depending on theexpression vector used and the transfection technique used. To identifyand select these integrants, a gene coding for a selectable marker (suchas the ones described above) is usually introduced into the host cellstogether with the gene of interest. These markers can for example beused in mutants in which these genes are not functional by, for example,deletion by conventional methods. Furthermore, nucleic acid moleculesencoding a selectable marker can be introduced into a host cell on thesame vector that comprises the sequence encoding the polypeptides of theinvention or used in the methods of the invention, or else in a separatevector. Cells which have been stably transfected with the introducednucleic acid can be identified for example by selection (for example,cells which have integrated the selectable marker survive whereas theother cells die). The marker genes may be removed or excised from thetransgenic cell once they are no longer needed. Techniques for markergene removal are known in the art, useful techniques are described abovein the definitions section.

Since the marker genes, particularly genes for resistance to antibioticsand herbicides, are no longer required or are undesired in thetransgenic host cell once the nucleic acids have been introducedsuccessfully, the process according to the invention for introducing thenucleic acids advantageously employs techniques which enable the removalor excision of these marker genes. One such a method is what is known asco-transformation. The co-transformation method employs two vectorssimultaneously for the transformation, one vector bearing the nucleicacid according to the invention and a second bearing the marker gene(s).A large proportion of transformants receives or, in the case of plants,comprises (up to 40% or more of the transformants), both vectors. Incase of transformation with Agrobacteria, the transformants usuallyreceive only a part of the vector, i.e. the sequence flanked by theT-DNA, which usually represents the expression cassette. The markergenes can subsequently be removed from the transformed plant byperforming crosses. In another method, marker genes integrated into atransposon are used for the transformation together with desired nucleicacid (known as the Ac/Ds technology). The transformants can be crossedwith a transposase source or the transformants are transformed with anucleic acid construct conferring expression of a transposase,transiently or stable. In some cases (approx. 10%), the transposon jumpsout of the genome of the host cell once transformation has taken placesuccessfully and is lost. In a further number of cases, the transposonjumps to a different location. In these cases the marker gene must beeliminated by performing crosses. In microbiology, techniques weredeveloped which make possible, or facilitate, the detection of suchevents. A further advantageous method relies on what is known asrecombination systems; whose advantage is that elimination by crossingcan be dispensed with. The best-known system of this type is what isknown as the Cre/lox system. Cre1 is a recombinase that removes thesequences located between the loxP sequences. If the marker gene isintegrated between the loxP sequences, it is removed once transformationhas taken place successfully, by expression of the recombinase. Furtherrecombination systems are the HIN/HIX, FLP/FRT and REP/STB system(Tribble et al., J. Biol. Chem., 275, 2000: 22255-22267; Velmurugan etal., J. Cell Biol., 149, 2000: 553-566). A site-specific integrationinto the plant genome of the nucleic acid sequences according to theinvention is possible. Naturally, these methods can also be applied tomicroorganisms such as yeast, fungi or bacteria.

Transgenic/Transgene/Recombinant

For the purposes of the invention, “transgenic”, “transgene” or“recombinant” means with regard to, for example, a nucleic acidsequence, an expression cassette, gene construct or a vector comprisingthe nucleic acid sequence or an organism transformed with the nucleicacid sequences, expression cassettes or vectors according to theinvention, all those constructions brought about by recombinant methodsin which either

-   -   (a) the nucleic acid sequences encoding proteins useful in the        methods of the invention, or    -   (b) genetic control sequence(s) which is operably linked with        the nucleic acid sequence according to the invention, for        example a promoter, or    -   (c) a) and b)        are not located in their natural genetic environment or have        been modified by recombinant methods, it being possible for the        modification to take the form of, for example, a substitution,        addition, deletion, inversion or insertion of one or more        nucleotide residues. The natural genetic environment is        understood as meaning the natural genomic or chromosomal locus        in the original plant or the presence in a genomic library. In        the case of a genomic library, the natural genetic environment        of the nucleic acid sequence is preferably retained, at least in        part. The environment flanks the nucleic acid sequence at least        on one side and has a sequence length of at least 50 bp,        preferably at least 500 bp, especially preferably at least 1000        bp, most preferably at least 5000 bp. A naturally occurring        expression cassette—for example the naturally occurring        combination of the natural promoter of the nucleic acid        sequences with the corresponding nucleic acid sequence encoding        a polypeptide useful in the methods of the present invention, as        defined above—becomes a transgenic expression cassette when this        expression cassette is modified by non-natural, synthetic        (“artificial”) methods such as, for example, mutagenic        treatment. Suitable methods are described, for example, in U.S.        Pat. No. 5,565,350 or WO 00/15815.

A transgenic plant for the purposes of the invention is thus understoodas meaning, as above, that the nucleic acids used in the method of theinvention are not at their natural locus in the genome of said plant, itbeing possible for the nucleic acids to be expressed homologously orheterologously. However, as mentioned, transgenic also means that, whilethe nucleic acids according to the invention or used in the inventivemethod are at their natural position in the genome of a plant, thesequence has been modified with regard to the natural sequence, and/orthat the regulatory sequences of the natural sequences have beenmodified. Transgenic is preferably understood as meaning the expressionof the nucleic acids according to the invention at an unnatural locus inthe genome, i.e. homologous or, preferably, heterologous expression ofthe nucleic acids takes place. Preferred transgenic plants are mentionedherein.

Transformation

The term “introduction” or “transformation” as referred to hereinencompasses the transfer of an exogenous polynucleotide into a hostcell, irrespective of the method used for transfer. Plant tissue capableof subsequent clonal propagation, whether by organogenesis orembryogenesis, may be transformed with a genetic construct of thepresent invention and a whole plant regenerated there from. Theparticular tissue chosen will vary depending on the clonal propagationsystems available for, and best suited to, the particular species beingtransformed. Exemplary tissue targets include leaf disks, pollen,embryos, cotyledons, hypocotyls, megagametophytes, callus tissue,existing meristematic tissue (e.g., apical meristem, axillary buds, androot meristems), and induced meristem tissue (e.g., cotyledon meristemand hypocotyl meristem). The polynucleotide may be transiently or stablyintroduced into a host cell and may be maintained non-integrated, forexample, as a plasmid. Alternatively, it may be integrated into the hostgenome. The resulting transformed plant cell may then be used toregenerate a transformed plant in a manner known to persons skilled inthe art.

The transfer of foreign genes into the genome of a plant is calledtransformation. Transformation of plant species is now a fairly routinetechnique. Advantageously, any of several transformation methods may beused to introduce the gene of interest into a suitable ancestor cell.The methods described for the transformation and regeneration of plantsfrom plant tissues or plant cells may be utilized for transient or forstable transformation. Transformation methods include the use ofliposomes, electroporation, chemicals that increase free DNA uptake,injection of the DNA directly into the plant, particle gun bombardment,transformation using viruses or pollen and microprojection. Methods maybe selected from the calcium/polyethylene glycol method for protoplasts(Krens, F. A. et al., (1982) Nature 296, 72-74; Negrutiu I et al. (1987)Plant Mol Biol 8: 363-373); electroporation of protoplasts (Shillito R.D. et al. (1985) Bio/Technol 3, 1099-1102); microinjection into plantmaterial (Crossway A et al., (1986) Mol. Gen Genet 202: 179-185); DNA orRNA-coated particle bombardment (Klein T M et al., (1987) Nature 327:70) infection with (non-integrative) viruses and the like. Transgenicplants, including transgenic crop plants, are preferably produced viaAgrobacterium-mediated transformation. An advantageous transformationmethod is the transformation in planta. To this end, it is possible, forexample, to allow the agrobacteria to act on plant seeds or to inoculatethe plant meristem with agrobacteria. It has proved particularlyexpedient in accordance with the invention to allow a suspension oftransformed agrobacteria to act on the intact plant or at least on theflower primordia. The plant is subsequently grown on until the seeds ofthe treated plant are obtained (Clough and Bent, Plant J. (1998) 16,735-743). Methods for Agrobacterium-mediated transformation of riceinclude well known methods for rice transformation, such as thosedescribed in any of the following: European patent application EP1198985 A1, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al.(Plant Mol Biol 22 (3): 491-506, 1993), Hiei et al. (Plant J 6 (2):271-282, 1994), which disclosures are incorporated by reference hereinas if fully set forth. In the case of corn transformation, the preferredmethod is as described in either Ishida et al. (Nat. Biotechnol 14(6):745-50, 1996) or Frame et al. (Plant Physiol 129(1): 13-22, 2002), whichdisclosures are incorporated by reference herein as if fully set forth.Said methods are further described by way of example in B. Jenes et al.,Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineeringand Utilization, eds. S. D. Kung and R. Wu, Academic Press (1993)128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42(1991) 205-225). The nucleic acids or the construct to be expressed ispreferably cloned into a vector, which is suitable for transformingAgrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. AcidsRes. 12 (1984) 8711). Agrobacteria transformed by such a vector can thenbe used in known manner for the transformation of plants, such as plantsused as a model, like Arabidopsis (Arabidopsis thaliana is within thescope of the present invention not considered as a crop plant), or cropplants such as, by way of example, tobacco plants, for example byimmersing bruised leaves or chopped leaves in an agrobacterial solutionand then culturing them in suitable media. The transformation of plantsby means of Agrobacterium tumefaciens is described, for example, byHofgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is knowninter alia from F. F. White, Vectors for Gene Transfer in Higher Plants;in Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D.Kung and R. Wu, Academic Press, 1993, pp. 15-38.

In addition to the transformation of somatic cells, which then have tobe regenerated into intact plants, it is also possible to transform thecells of plant meristems and in particular those cells which developinto gametes. In this case, the transformed gametes follow the naturalplant development, giving rise to transgenic plants. Thus, for example,seeds of Arabidopsis are treated with agrobacteria and seeds areobtained from the developing plants of which a certain proportion istransformed and thus transgenic [Feldman, K A and Marks M D (1987). MolGen Genet 208:274-289; Feldmann K (1992). In: C Koncz, N-H Chua and JShell, eds, Methods in Arabidopsis Research. Word Scientific, Singapore,pp. 274-289]. Alternative methods are based on the repeated removal ofthe inflorescences and incubation of the excision site in the center ofthe rosette with transformed agrobacteria, whereby transformed seeds canlikewise be obtained at a later point in time (Chang (1994). Plant J. 5:551-558; Katavic (1994). Mol Gen Genet, 245: 363-370). However, anespecially effective method is the vacuum infiltration method with itsmodifications such as the “floral dip” method. In the case of vacuuminfiltration of Arabidopsis, intact plants under reduced pressure aretreated with an agrobacterial suspension [Bechthold, N (1993). C R AcadSci Paris Life Sci, 316: 1194-1199], while in the case of the “floraldip” method the developing floral tissue is incubated briefly with asurfactant-treated agrobacterial suspension [Clough, S J and Bent A F(1998) The Plant J. 16, 735-743]. A certain proportion of transgenicseeds are harvested in both cases, and these seeds can be distinguishedfrom non-transgenic seeds by growing under the above-described selectiveconditions. In addition the stable transformation of plastids is ofadvantages because plastids are inherited maternally is most cropsreducing or eliminating the risk of transgene flow through pollen. Thetransformation of the chloroplast genome is generally achieved by aprocess which has been schematically displayed in Klaus et al., 2004[Nature Biotechnology 22 (2), 225-229]. Briefly the sequences to betransformed are cloned together with a selectable marker gene betweenflanking sequences homologous to the chloroplast genome. Thesehomologous flanking sequences direct site specific integration into theplastome. Plastidal transformation has been described for many differentplant species and an overview is given in Bock (2001) Transgenicplastids in basic research and plant biotechnology. J Mol Biol. 2001Sep. 21; 312 (3):425-38 or Maliga, P (2003) Progress towardscommercialization of plastid transformation technology. TrendsBiotechnol. 21, 20-28. Further biotechnological progress has recentlybeen reported in form of marker free plastid transformants, which can beproduced by a transient co-integrated maker gene (Klaus et al., 2004,Nature Biotechnology 22(2), 225-229).

T-DNA Activation Taming

T-DNA activation tagging (Hayashi et al. Science (1992) 1350-1353),involves insertion of T-DNA, usually containing a promoter (may also bea translation enhancer or an intron), in the genomic region of the geneof interest or 10 kb up- or downstream of the coding region of a gene ina configuration such that the promoter directs expression of thetargeted gene. Typically, regulation of expression of the targeted geneby its natural promoter is disrupted and the gene falls under thecontrol of the newly introduced promoter. The promoter is typicallyembedded in a T-DNA. This T-DNA is randomly inserted into the plantgenome, for example, through Agrobacterium infection and leads tomodified expression of genes near the inserted T-DNA. The resultingtransgenic plants show dominant phenotypes due to modified expression ofgenes close to the introduced promoter.

TILLING

The term “TILLING” is an abbreviation of “Targeted Induced Local LesionsIn Genomes” and refers to a mutagenesis technology useful to generateand/or identify nucleic acids encoding proteins with modified expressionand/or activity. TILLING also allows selection of plants carrying suchmutant variants. These mutant variants may exhibit modified expression,either in strength or in location or in timing (if the mutations affectthe promoter for example). These mutant variants may exhibit higheractivity than that exhibited by the gene in its natural form. TILLINGcombines high-density mutagenesis with high-throughput screeningmethods. The steps typically followed in TILLING are: (a) EMSmutagenesis (Redei G P and Koncz C (1992) In Methods in ArabidopsisResearch, Koncz C, Chua N H, Schell J, eds. Singapore, World ScientificPublishing Co, pp. 16-82; Feldmann et al., (1994) In Meyerowitz E M,Somerville C R, eds, Arabidopsis. Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., pp 137-172; Lightner J and Caspar T (1998) InJ Martinez-Zapater, J Salinas, eds, Methods on Molecular Biology, Vol.82. Humana Press, Totowa, N.J., pp 91-104); (b) DNA preparation andpooling of individuals; (c) PCR amplification of a region of interest;(d) denaturation and annealing to allow formation of heteroduplexes; (e)DHPLC, where the presence of a heteroduplex in a pool is detected as anextra peak in the chromatogram; (f) identification of the mutantindividual; and (g) sequencing of the mutant PCR product. Methods forTILLING are well known in the art (McCallum et al., (2000) NatBiotechnol 18: 455-457; reviewed by Stemple (2004) Nat Rev Genet 5(2):145-50).

Homologous Recombination

Homologous recombination allows introduction in a genome of a selectednucleic acid at a defined selected position. Homologous recombination isa standard technology used routinely in biological sciences for lowerorganisms such as yeast or the moss Physcomitrella. Methods forperforming homologous recombination in plants have been described notonly for model plants (Offringa et al. (1990) EMBO J 9(10): 3077-84) butalso for crop plants, for example rice (Terada et al. (2002) Nat Biotech20(10): 1030-4; Iida and Terada (2004) Curr Opin Biotech 15(2): 132-8),and approaches exist that are generally applicable regardless of thetarget organism (Miller et al, Nature Biotechnol. 25, 778-785, 2007).

Yield

The term “yield” in general means a measurable produce of economicvalue, typically related to a specified crop, to an area, and to aperiod of time. Individual plant parts directly contribute to yieldbased on their number, size and/or weight, or the actual yield is theyield per square meter for a crop and year, which is determined bydividing total production (includes both harvested and appraisedproduction) by planted square meters. The term “yield” of a plant mayrelate to vegetative biomass (root and/or shoot biomass), toreproductive organs, and/or to propagules (such as seeds) of that plant.

Early Vigour

“Early vigour” refers to active healthy well-balanced growth especiallyduring early stages of plant growth, and may result from increased plantfitness due to, for example, the plants being better adapted to theirenvironment (i.e. optimizing the use of energy resources andpartitioning between shoot and root). Plants having early vigour alsoshow increased seedling survival and a better establishment of the crop,which often results in highly uniform fields (with the crop growing inuniform manner, i.e. with the majority of plants reaching the variousstages of development at substantially the same time), and often betterand higher yield. Therefore, early vigour may be determined by measuringvarious factors, such as thousand kernel weight, percentage germination,percentage emergence, seedling growth, seedling height, root length,root and shoot biomass and many more.

Increase/Improve/Enhance

The terms “increase”, “improve” or “enhance” are interchangeable andshall mean in the sense of the application at least a 3%, 4%, 5%, 6%,7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%,30%, 35% or 40% more yield and/or growth in comparison to control plantsas defined herein.

Seed Yield

Increased seed yield may manifest itself as one or more of thefollowing: a) an increase in seed biomass (total seed weight) which maybe on an individual seed basis and/or per plant and/or per square meter;b) increased number of flowers per plant; c) increased number of(filled) seeds; d) increased seed filling rate (which is expressed asthe ratio between the number of filled seeds divided by the total numberof seeds); e) increased harvest index, which is expressed as a ratio ofthe yield of harvestable parts, such as seeds, divided by the totalbiomass; and f) increased thousand kernel weight (TKW), and g) increasednumber of primary panicles, which is extrapolated from the number offilled seeds counted and their total weight. An increased TKW may resultfrom an increased seed size and/or seed weight, and may also result froman increase in embryo and/or endosperm size.

An increase in seed yield may also be manifested as an increase in seedsize and/or seed volume. Furthermore, an increase in seed yield may alsomanifest itself as an increase in seed area and/or seed length and/orseed width and/or seed perimeter. Increased seed yield may also resultin modified architecture, or may occur because of modified architecture.

Greenness Index

The “greenness index” as used herein is calculated from digital imagesof plants. For each pixel belonging to the plant object on the image,the ratio of the green value versus the red value (in the RGB model forencoding color) is calculated. The greenness index is expressed as thepercentage of pixels for which the green-to-red ratio exceeds a giventhreshold. Under normal growth conditions, under salt stress growthconditions, and under reduced nutrient availability growth conditions,the greenness index of plants is measured in the last imaging beforeflowering. In contrast, under drought stress growth conditions, thegreenness index of plants is measured in the first imaging afterdrought.

Plant

The term “plant” as used herein encompasses whole plants, ancestors andprogeny of the plants and plant parts, including seeds, shoots, stems,leaves, roots (including tubers), flowers, and tissues and organs,wherein each of the aforementioned comprise the gene/nucleic acid ofinterest. The term “plant” also encompasses plant cells, suspensioncultures, callus tissue, embryos, meristematic regions, gametophytes,sporophytes, pollen and microspores, again wherein each of theaforementioned comprises the gene/nucleic acid of interest.

Plants that are particularly useful in the methods of the inventioninclude all plants which belong to the superfamily Viridiplantae, inparticular monocotyledonous and dicotyledonous plants including fodderor forage legumes, ornamental plants, food crops, trees or shrubsselected from the list comprising Acer spp., Actinidia spp., Abelmoschusspp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp.,Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apiumgraveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avenaspp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var.sativa, Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasahispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g.Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape]),Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa,Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Caryaspp., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichoriumendivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp.,Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., Coriandrumsativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp.,Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpuslongan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g.Elaeis guineensis, Elaeis oleifera), Eleusine coracana, Eragrostis tef,Erianthus sp., Eriobotrya japonica, Eucalyptus sp., Eugenia uniflora,Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficus carica,Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g.Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthusspp. (e.g. Helianthus annuus), Hemerocallis fulva, Hibiscus spp.,Hordeum spp. (e.g. Hordeum vulgare), Ipomoea batatas, Juglans spp.,Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum,Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzulasylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersiconlycopersicum, Lycopersicon pyriforme), Macrotyloma spp., Malus spp.,Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp.,Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp.,Miscanthus sinensis, Momordica spp., Morus nigra, Musa spp., Nicotianaspp., Olea spp., Opuntia spp., Omithopus spp., Oryza spp. (e.g. Oryzasativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum,Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp.,Petroselinum crispum, Phalaris arundinacea, Phaseolus spp., Phleumpratense, Phoenix spp., Phragmites australis, Physalis spp., Pinus spp.,Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunusspp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp.,Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubusspp., Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamumspp., Sinapis sp., Solanum spp. (e.g. Solanum tuberosum, Solanumintegrifolium or Solanum lycopersicum), Sorghum bicolor, Spinacia spp.,Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao,Trifolium spp., Tripsacum dactyloides, Triticale sp., Triticosecalerimpaui, Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticumturgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticummonococcum or Triticum vulgare), Tropaeolum minus, Tropaeolum majus,Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zeamays, Zizania palustris, Ziziphus spp., amongst others.

DETAILED DESCRIPTION OF THE INVENTION

Surprisingly, it has now been found that modulating expression in aplant of a nucleic acid encoding a RHL1 polypeptide gives plants havingenhanced yield-related traits relative to control plants. According to afirst embodiment, the present invention provides a method for enhancingyield-related traits in plants relative to control plants, comprisingmodulating expression in a plant of a nucleic acid encoding a RHL1polypeptide.

Furthermore, surprisingly, it has now been found that increasingexpression in a plant of a nucleic acid sequence encoding a TGasepolypeptide as defined herein, gives plants having increased seedyield-related traits relative to control plants. According to a firstembodiment, the present invention provides a method for increasing seedyield-related traits in plants relative to control plants, comprisingincreasing expression in a plant of a nucleic acid sequence encoding aTGase polypeptide.

Furthermore, surprisingly, it has now been found that modulatingexpression in a plant of a nucleic acid encoding a TRY-like polypeptidegives plants having enhanced yield-related traits relative to controlplants. According to a first embodiment, the present invention providesa method for enhancing yield-related traits in plants relative tocontrol plants, comprising modulating expression in a plant of a nucleicacid encoding a TRY-like polypeptide and optionally selecting for plantshaving enhanced yield-related traits.

Furthermore, surprisingly, it has now been found that modulatingexpression in a plant of a nucleic acid encoding a BZR polypeptide givesplants having increased seed yield relative to control plants. Accordingto a first embodiment, the present invention provides a method forincreasing seed yield in plants relative to control plants, comprisingmodulating expression in a plant of a nucleic acid encoding a BZRpolypeptide.

A preferred method for modulating (preferably, increasing) expression ofa nucleic acid sequence encoding a RHL1 polypeptide, or a TGasepolypeptide, or a TRY-like polypeptide, or a BZR polypeptide, is byintroducing and expressing in a plant a nucleic acid sequence encoding aRHL1 polypeptide, or a TGase polypeptide, or a TRY-like polypeptide, ora BZR polypeptide.

Concerning BZR polypeptides, in a further preferred embodiment theinvention provides a method for increasing seed yield in plants relativeto control plants, comprising modulating expression in a plant of anucleic acid encoding a BZR polypeptide wherein said modulation iseffected by introducing a nucleic acid encoding a BZR polypeptide underthe control of a plant derived promoter.

Concerning RHL1 polypeptides, any reference hereinafter to a “proteinuseful in the methods of the invention” is taken to mean a RHL1polypeptide as defined herein. Any reference hereinafter to a “nucleicacid useful in the methods of the invention” is taken to mean a nucleicacid capable of encoding such a RHL1 polypeptide. The nucleic acid to beintroduced into a plant (and therefore useful in performing the methodsof the invention) is any nucleic acid encoding the type of protein whichwill now be described, hereafter also named “RHL1 nucleic acid” or “RHL1gene”.

An RHL1 polypeptide” as defined herein refers to any polypeptidecomprising a sequence having in increasing order of preference 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or more sequence identity to the amino acid sequence ofany of the polypeptides of Table A1.

A preferred RHL1 polypeptide useful in the methods of the inventioncomprises a sequence having in increasing order of preference 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or moresequence identity to the amino acid sequence of any of the polypeptidesof SEQ ID NO: 2 or SEQ ID NO: 10, more preferably comprises SEQ ID NO:2.

Various conserved protein motifs are found RHL1 polypeptides. Methods tofind conserved protein domain in a group of related sequences are wellknown in the art. Example 4 details the use of one such method, the MEMEsystem, to identify conserved protein motifs in RHL1 polypeptides.

A further preferred RHL1 polypeptide useful in the methods of theinvention comprises one or more of the following motifs:

-   -   (i) a motif having in increasing order of preference 50%, 55%,        60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or        more sequence identity to the amino acid sequence of Motif 1:        [IV]R[RK][KG][SG]QRK[NS][RK][FY]L FSFPGLLAP (SEQ ID NO: 29);    -   (ii) a motif having in increasing order of preference 50%, 55%,        60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or        more sequence identity to the amino acid sequence of Motif 2:        SGG[KR][IV]G[ED]L[KA]DL[GD]TKNP[ILV]LYLDFPQG[RQ]MKL](SEQ ID NO:        30);    -   (iii) a motif having in increasing order of preference 50%, 55%,        60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or        more sequence identity to the amino acid sequence of Motif 3:        TP[VS]RQSARTAGKK[FL][KN][FY][AT]ExSS (SEQ ID NO: 31);    -   (iv) a motif having in increasing order of preference 50%, 55%,        60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or        more sequence identity to the amino acid sequence of Motif 4:        GTK[ED]ENPEE[LA][RK]L[DE]FPKE[LF]Q[ENQ][GD](SEQ ID NO: 32);    -   (v) a motif having in increasing order of preference 50%, 55%,        60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or        more sequence identity to the amino acid sequence of Motif        5:[SN][GN][NL]L[LQV][SR][EDG]xP[AS][KA]PR[SA][APS]LAPSK[TAG]VL[KR][HL][HQ]G[KR]D        (SEQ ID NO: 33);    -   (vi) a motif having in increasing order of preference 50%, 55%,        60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or        more sequence identity to the amino acid sequence of Motif 6:        HA[ED][CY]DFKGGAGAA[CS]D[ES][KA]Q (SEQ ID NO: 34);    -   (vii) a motif having in increasing order of preference 50%, 55%,        60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or        more sequence identity to the amino acid sequence of Motif        7:[KSN][KEP]P[GEK][EKT][KTE][YT][VT][EG][EPST][ELQ]SP[KE][IT][ED][SLV][ED][DI][DV][LS]S[ED][DE][SD][NDS][LD]K[DK](SEQ        ID NO: 35);    -   (viii) a motif having in increasing order of preference 50%,        55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%        or more sequence identity to the amino acid sequence of Motif 8:        KG[PA]AAKKQRASP[EM][EA]K[HQ]P[TA]G[KI]K (SEQ ID NO: 36).

Wherein the amino acids between square brackets are alternatives.

Alternatively a preferred RHL1 polypeptide useful in the methods of theinvention comprises:

A. a motif having in increasing order of preference 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to the amino acid sequence of one or more of the followingmotifs:

(i) Motif 9: (SEQ ID NO: 37)(SN)VMC(ED)D(YV)F(DE)(NS)(ML)(IV)VFS(DE)AWWIG(TR) K(ED)ENPEE;(ii) Motif 10: (SEQ ID NO: 38)L(AILV)A(PA)(IVA)(SA)GG(KR)(IVF)G(ED)L(KA)DL(GDS) (TS)KNP(IVL)LYLDFPQ;(iii) Motif 11: (SEQ ID NO: 39) G(RQ)(ML)KLFGTI(VL)YPKN(RK)Y(LI)TLQF;Wherein the amino acids between brackets (alternative amino acids atthat position), are alternatives; orB. any one or more of the following motifs:

(i) Motif 9: (SEQ ID NO: 37)(SN)VMC(ED)D(YV)F(DE)(NS)(ML)(IV)VFS(DE)AWWIG(TR) K(ED)ENPEE;(ii) Motif 10: (SEQ ID NO: 38)L(AILV)A(PA)(IVA)(SA)GG(KR)(IVF)G(ED)L(KA)DL(GDS) (TS)KNP(IVL)LYLDFPQ;(iii) Motif 11: (SEQ ID NO: 39) G(RQ)(ML)KLFGTI(VL)YPKN(RK)Y(LI)TLQF;

Wherein the amino acids between brackets are alternatives (alternativeamino acids at that position), and wherein in increasing order ofpreference 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids are substitutedby any other amino acid, preferably by a conservative amino acid.

An even further preferred RHL1 polypeptides useful in the methods of theinvention are paralogous or orthologous proteins of any of thepolypeptides of Table A.

Alternatively, the homologue of an RHL1 protein has in increasing orderof preference at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%,49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identityto the amino acid represented by SEQ ID NO: 2, provided that thehomologous protein comprises one of the conserved motifs as outlinedabove. The overall sequence identity is determined using a globalalignment algorithm, such as the Needleman Wunsch algorithm in theprogram GAP (GCG Wisconsin Package, Accelrys), preferably with defaultparameters. Compared to overall sequence identity, the sequence identitywill generally be higher when only conserved domains or motifs areconsidered.

Preferably, the polypeptide sequence which when used in the constructionof a phylogenetic tree, such as the one depicted in FIG. 3, clusterswith any of the RHL1 polypeptides originating from a dicotyledoneousplant comprising the amino acid sequence represented by SEQ ID NO: 2rather than with any other group.

Concerning TGase, any reference hereinafter to a “protein useful in themethods of the invention” is taken to mean a TGase polypeptide asdefined herein. Any reference hereinafter to a “nucleic acid sequenceuseful in the methods of the invention” is taken to mean a nucleic acidsequence capable of encoding such a TGase polypeptide. The nucleic acidsequence to be introduced into a plant (and therefore useful inperforming the methods of the invention) is any nucleic acid sequenceencoding the type of polypeptide, which will now be described, hereafteralso named “TGase nucleic acid sequence” or “TGase gene”.

A “TGase polypeptide” as defined herein refers to any polypeptidecomprising (i) a plastidic transit peptide; (ii) in increasing order ofpreference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, 99% or more amino acid sequence identity to a domain comprising atleast one coiled coil as represented by SEQ ID NO: 70; (iii) and anIntegrated relational Enzyme database entry EC 2.3.2.13 forprotein-glutamine γ-glutamyltransferase.

Alternatively or additionally, a “TGase polypeptide” as defined hereinrefers to any polypeptide sequence having (i) a plastidic transitpeptide; (ii) in increasing order of preference at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequenceidentity to a polypeptide as represented by SEQ ID NO: 45.

Alternatively or additionally, a “TGase polypeptide” as defined hereinrefers to any polypeptide having in increasing order of preference atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or moreamino acid sequence identity to a TGase polypeptide as represented bySEQ ID NO: 45, or to any of the polypeptide sequences given in Table A2herein.

Alternatively or additionally, a “TGase polypeptide” as defined hereinrefers to any polypeptide sequence which when used in the constructionof a TGase phylogenetic tree, such as the one depicted in FIG. 5,clusters with the clade of TGase polypeptides comprising the polypeptidesequence as represented by SEQ ID NO: 45 (marked by an arrow in FIG. 5;TGases from plants are delimited by a bracket in FIG. 5), rather thanwith the other clades.

Alternatively or additionally, a “TGase polypeptide” is a polypeptidewith enzymatic activity consisting in catalyzing the formation of amidelinkages, generally in a Ca-dependent fashion, between the primary amineof an amine donor substrate and the y-carboxamide group of peptide-boundendo-glutamine residues in proteins or polypeptides that are the amineacceptors.

Concerning ny reference hereinafter to a “protein useful in the methodsof the invention” is taken to mean a TRY-like polypeptide as definedherein. Any reference hereinafter to a “nucleic acid useful in themethods of the invention” is taken to mean a nucleic acid capable ofencoding such a TRY-like polypeptide. The nucleic acid to be introducedinto a plant (and therefore useful in performing the methods of theinvention) is any nucleic acid encoding the type of protein which willnow be described, hereafter also named “TRY-like nucleic acid” or“TRY-like gene”.

A “TRY-like polypeptide” as defined herein refers to any polypeptidecomprising a Myb-like DNA-binding domain (PFam domain PF00249.17, SMARTdomain SM00717, ProfileScan domain PS50090, Panther PTHR10641:SF26).

Preferably, the TRY-like polypeptide comprises one or more of thefollowing motifs:

Motif 12: [FM][ST]E[DQ]EE[DT]LIIRM[YHF][NKR]LVG[EDN]RW[SE] LIAGRIMotif 13: PGR[TK]AEEIE[KR][YF]WT[SM][RK] Motif 14:EEVSS[QT][ED][SW][EK][FL][IE] Motif 15:E[ED][DET][LI][IV]X[RK][LFM][HY]XL[LFV]G[NED][RK]WX[LI]I A[GRK]R[LIV][PV]GR[TKEQ][DAP][NEKG][EQ] [IVQ]wherein X on position 6 may be any amino acid, but preferably one of I,V, L, Y, S, F, C, or T; and wherein X on position 10 may be any aminoacid, but preferably one of R, K, E, S, T, or N; and wherein X onposition 17 may be any amino acid, but preferably one of S, D, A, E, P,or T. Preferably Motif 15 isEE[DT][LI][IV]XRM[HY][RKN]LVG[NED]RWX[LI]IA[GR]R[IV][PV]GR[TKEQ][AP][NEKG]E[IVQ]wherein X on position 6 may be any amino acid, but preferably one of Y,S, F, C, or T; and wherein X on position 17 may be any amino acid, butpreferably one of D, A, E, P, or T.

More preferably, the TRY-like polypeptide comprises in increasing orderof preference, at least 2, at least 3, or all 4 motifs.

Alternatively, the homologue of a TRY-like protein has in increasingorder of preference at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overallsequence identity to the amino acid represented by SEQ ID NO: 76,provided that the homologous protein comprises the conserved motifs asoutlined above. The overall sequence identity is determined using aglobal alignment algorithm, such as the Needleman Wunsch algorithm inthe program GAP (GCG Wisconsin Package, Accelrys), preferably withdefault parameters and preferably with sequences of mature proteins(i.e. without taking into account secretion signals or transitpeptides). Compared to overall sequence identity, the sequence identitywill generally be higher when only conserved domains or motifs areconsidered. Preferably the motifs in a TRY-like polypeptide have, inincreasing order of preference, at least 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity tothe motifs represented by SEQ ID NO: 229 to SEQ ID NO: 232 (Motifs 12 to15).

Preferably, the polypeptide sequence which when used in the constructionof a phylogenetic tree, constructed with the polypeptide sequences ofTable A3, clusters with the group of TRY-like polypeptides comprisingthe amino acid sequence represented by SEQ ID NO: 76 (At5g53200) ratherthan with any other group.

Concerning BZR polypeptides, any reference hereinafter to a “proteinuseful in the methods of the invention” is taken to mean a BZRpolypeptide as defined herein. Any reference hereinafter to a “nucleicacid useful in the methods of the invention” is taken to mean a nucleicacid capable of encoding such a BZR polypeptide. The nucleic acid to beintroduced into a plant (and therefore useful in performing the methodsof the invention) is any nucleic acid encoding the type of protein whichwill now be described, hereafter also named “BZR nucleic acid” or “BZRgene”.

A “BZR polypeptide” as defined herein refers to any transcription factorpolypeptide comprising a BZR1 transcriptional repressor domain (Interproaccession number: IPR008540). Typically the N-terminus of BZRpolypetides comprises one or more nuclear localization signals and abHLH-like DNA binding domain (Yin et al. (2008) Plant Physiology147:280-295.

BZR transcription factors are well known in the art. BZR polypeptidesbelong to a small family of proteins of plant origin which function astranscriptional modulators involved in controlling the response toBrassinosteroids (BRs).

The BZR polypeptide useful in the methods of the invention comprises adomain having in increasing order of preference at least 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to the BZR1 transcriptionalrepressor domain in SEQ ID NO: 238 located at amino acid position(coordinates) 10 to 157 in SEQ ID NO: 238 or to a BZR transcriptionalrepressor domain comprised in any of the polypeptides of Table A4.

Typically, the BZR polypeptides useful in the methods of the inventionhave a conserved bHLH-like domain located at the N-terminus of theprotein for example such domain corresponds to the sequenceRERRRRAIAAKIFTGLRSQGNYKLPKHCDNNEVLKALCLE AGWIVHEDGT: (SEQ ID NO: 326)located at positions 27 to 76 of SEQ ID NO: 238.

Additionally the BZR polypeptide useful in the methods of the inventionmay comprise a domain having in increasing order of preference at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to abHLH-like domain as represented by SEQ ID NO: 326.

Additionally, the BZR polypeptide useful in the methods of the inventionmay comprise any one or more of the following motifs:

-   -   (i) Motif 16: SAPVTPPLSSP (SEQ ID NO: 323), wherein 1, 2, 3 or 4        residues may be substituted by any amino acid.    -   (ii) Motif 17: VKPWEGERIHE (SEQ ID NO: 324), wherein 1, 2, 3 or        4 residues may be substituted by any amino acid.    -   (iii) Motif 18: DLELTLG (SEQ ID NO: 325), wherein 1, 2, 3 or 4        residues may be substituted by any amino acid.

Alternatively, the homologue of a BZR protein has in increasing order ofpreference at least 20%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%,34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%,48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequenceidentity to the amino acid represented by SEQ ID NO: 238, provided thatthe homologous protein comprises the conserved BZR domain as outlinedabove. The overall sequence identity is determined using a globalalignment algorithm, such as the Needleman Wunsch algorithm in theprogram GAP (GCG Wisconsin Package, Accelrys), preferably with defaultparameters and preferably with sequences of mature proteins (i.e.without taking into account secretion signals or transit peptides).Compared to overall sequence identity, the sequence identity willgenerally be higher when only conserved domains or motifs areconsidered.

The terms “domain”, “signature” and “motif” are defined in the“definitions” section herein. Specialist databases exist for theidentification of domains, for example, SMART (Schultz et al. (1998)Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) NucleicAcids Res 30, 242-244), InterPro (Mulder et al., (2003) Nucl. Acids.Res. 31, 315-318), Prosite (Bucher and Bairoch (1994), A generalizedprofile syntax for biomolecular sequences motifs and its function inautomatic sequence interpretation. (In) ISMB-94; Proceedings 2ndInternational Conference on Intelligent Systems for Molecular Biology.Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp 53-61,AAAI Press, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137,(2004)), or Pfam (Bateman et al., Nucleic Acids Research 30(1): 276-280(2002)). A set of tools for in silico analysis of protein sequences isavailable on the ExPASy proteomics server (Swiss Institute ofBioinformatics (Gasteiger et al., ExPASy: the proteomics server forin-depth protein knowledge and analysis, Nucleic Acids Res.31:3784-3788(2003)). Domains or motifs may also be identified usingroutine techniques, such as by sequence alignment.

Concerning TGase plypeptides, the term “domain” and “motif” is definedin the “definitions” section herein. Specialist databases exist for theidentification of domains, for example, SMART (Schultz et al. (1998)Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) NucleicAcids Res 30, 242-244), InterPro (Mulder et al., (2003) Nucl. Acids.Res. 31, 315-318), Prosite (Bucher and Bairoch (1994), A generalizedprofile syntax for biomolecular sequences motifs and its function inautomatic sequence interpretation. (In) ISMB-94; Proceedings 2ndInternational Conference on Intelligent Systems for Molecular Biology.Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp 53-61,AAAI Press, Menlo Park; Hulo et al., Nucl. Acids. Res. 32: D134-D137,(2004)), or Pfam (Bateman et al., Nucleic Acids Research 30(1): 276-280(2002). A set of tools for in silico analysis of protein sequences isavailable on the ExPASy proteomics server (Swiss Institute ofBioinformatics (Gasteiger et al., ExPASy: the proteomics server forin-depth protein knowledge and analysis, Nucleic Acids Res.31:3784-3788(2003)). Domains may also be identified using routinetechniques, such as by sequence alignment. An alignment of thepolypeptides of Table A2 herein, is shown in FIG. 6. Such alignments areuseful for identifying the most conserved domains or motifs between theTGase polypeptides as defined herein. One such domain is a domaincomprising at least one coiled coil, marked by X's in FIG. 6, and asrepresented by SEQ ID NO: 70.

Concerning TGase polypeptides, coiled coils are domains that areimportant to identify for protein-protein interactions, such asoligomerization, either of identical proteins, of proteins of the samefamily, or of unrelated proteins. Recently much progress has been madein computational prediction of coiled coils from sequence data. Amongalgorithms well known to a person skilled in the art are available atthe ExPASy Proteomics tools COILS, PAIRCOIL, PAIRCOIL2, MULTICOIL, orMARCOIL, hosted by the Swiss Institute for Bioinformatics. In Example 4and FIG. 5, are shown respectively the numerical and graphical resultsof SEQ ID NO: 45 as produced by the COILS algorithm analysis. A domaincomprising at one coiled coil is identified in the TGase polypeptidesequence as represented by SEQ ID NO: 45, and is represented as in SEQID NO: 70.

Methods for the alignment of sequences for comparison are well known inthe art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAPuses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48:443-453) to find the global (i.e. spanning the complete sequences)alignment of two sequences that maximizes the number of matches andminimizes the number of gaps. The BLAST algorithm (Altschul et al.(1990) J Mol Biol 215: 403-10) calculates percent sequence identity andperforms a statistical analysis of the similarity between the twosequences. The software for performing BLAST analysis is publiclyavailable through the National Centre for Biotechnology Information(NCBI). Homologues may readily be identified using, for example, theClustalW multiple sequence alignment algorithm (version 1.83), with thedefault pairwise alignment parameters, and a scoring method inpercentage. Global percentages of similarity and identity may also bedetermined using one of the methods available in the MatGAT softwarepackage (Campanella et al., BMC Bioinformatics. 2003 Jul. 10; 4:29.MatGAT: an application that generates similarity/identity matrices usingprotein or DNA sequences.). Minor manual editing may be performed tooptimise alignment between conserved motifs, as would be apparent to aperson skilled in the art. Furthermore, instead of using full-lengthsequences for the identification of homologues, specific domains mayalso be used. The sequence identity values may be determined over theentire nucleic acid or amino acid sequence or over selected domains orconserved motif(s), using the programs mentioned above using the defaultparameters. For local alignments, the Smith-Waterman algorithm isparticularly useful (Smith T F, Waterman M S (1981) J. Mol. Biol 147(1);195-7).

Concerning TGase polypeptides, Example 3 herein describes in Table B2the percentage identity between the TGase polypeptide as represented bySEQ ID NO: 45 and the TGase polypeptides listed in Table A2, which canbe as low as 26% amino acid sequence identity.

The task of protein subcellular localisation prediction is important andwell studied. Knowing a protein's localisation helps elucidate itsfunction. Experimental methods for protein localization range fromimmunolocalization to tagging of proteins using green fluorescentprotein (GFP) or beta-glucuronidase (GUS). Such methods are accuratealthough labor-intensive compared with computational methods. Recentlymuch progress has been made in computational prediction of proteinlocalisation from sequence data. Among algorithms well known to a personskilled in the art are available at the ExPASy Proteomics tools hostedby the Swiss Institute for Bioinformatics, for example, PSort, TargetP,ChloroP, LocTree, Predotar, LipoP, MITOPROT, PATS, PTS1, SignalP, TMHMM,and others. The subcellular localisation of polypeptides useful inperforming the methods of the invention was previously described in theliterature (Villalobos et al. (2004) Gene 336: 93-104). In particularSEQ ID NO: 45 of the present invention is assigned to the plastidic(chloroplastic) compartment of plant cells.

Methods for targeting to plastids are well known in the art and includethe use of transit peptides. Table 3 below shows examples of transitpeptides which can be used to target any TGase polypeptide to a plastid,which TGase polypeptide is not, in its natural form, normally targetedto a plastid, or which TGase polypeptide in its natural form is targetedto a plastid by virtue of a different transit peptide (for example, itsnatural transit peptide). Cloning a nucleic acid sequence encoding atransit peptide upstream and in-frame of a nucleic acid sequenceencoding a polypeptide (for example, a TGase polypeptide lacking its owntransit peptide), involves standard molecular techniques that arewell-known in the art.

TABLE 3 Examples of transit peptide sequences useful in targetingpolypeptides to plastids NCBI Accession Number/ Source Protein SEQ ID NOOrganism Function Transit Peptide Sequence SEQ ID NO: ChlamydomonasFerredoxin MAMAMRSTFAARVGAKPAVRGARPAS P07839 RMSCMA SEQ ID NO:Chlamydomonas Rubisco MQVTMKSSAVSGQRVGGARVATRSVR AAR23425 activaseRAQLQV SEQ ID NO: Arabidopsis Aspartate aminoMASLMLSLGSTSLLPREINKDKLKLGTS CAA56932 thaliana transferaseASNPFLKAKSFSRVTMTVAVKPSR SEQ ID NO: Arabidopsis Acyl carrierMATQFSASVSLQTSCLATTRISFQKPAL CAA31991 thaliana protein1ISNHGKTNLSFNLRRSIPSRRLSVSC SEQ ID NO: Arabidopsis Acyl carrierMASIAASASISLQARPRQLAIAASQVKS CAB63798 thaliana protein2FSNGRRSSLSFNLRQLPTRLTVSCAAKP ETVDKVCAVVRKQL SEQ ID NO: ArabidopsisAcyl carrier MASIATSASTSLQARPRQLVIGAKQVKS CAB63799 thaliana protein3FSYGSRSNLSFNLRQLPTRLTVYCAAKP ETVDKVCAVVRKQLSLKE

The TGase polypeptide is targeted and active in the chloroplast, i.e.,the TGase polypeptide is capable of consisting in catalyzing theformation of amide linkages, generally in a Ca-dependent fashion,between the primary amine of an amine donor substrate and they-carboxamide group of peptide-bound endo-glutamine residues in proteinsor polypeptides that are the amine acceptors (Villalobos et al. (2004)Gene 336: 93-104).

Furthermore, RHL1 polypeptides typically have DNA biding activity. Toolsand techniques for measuring DNA biding activity are well known in theart. Further details are provided in the Examples section.

In addition, RHL1 polypeptides, when expressed in rice according to themethods of the present invention as outlined in the Examples section,give plants having increased yield related traits, in particular any oneof early vigour, increased total seed weight per plant, increased numberof seeds, increased number of filled seeds and increased harvest index.

Furthermore, TRY-like polypeptides (at least in their native form)typically have DNA binding activity. Tools and techniques for measuringDNA binding activity are well known in the art.

In addition, TRY-like polypeptides, when expressed in rice according tothe methods of the present invention as outlined in the Examplessection, give plants having increased yield related traits, inparticular one or more of increased emergence vigour, increased fillrate, increased harvest index, increased total number of seeds,increased thousand kernel weight, increased number of first panicles,increased number of filled seeds and/or increased total weight of seeds.

Furthermore, BZR polypeptides (at least in their native form) typicallyhave DNA-binding activity and optionally protein-binding activity. Toolsand techniques for measuring DNA binding activity are well known in theart. For example the EMSA technique which is based is based on theobservation that protein:DNA complexes migrate more slowly than free DNAmolecules when subjected to non-denaturing polyacrylamide or agarose gelelectrophoresis may be used (He et al. Science 307, 134-138 (2005)).Techniques useful to determine interaction between polypeptides are wellknown in the art and include but are not limited to yeast two hybrid,inmunoprecipation, or affinity purification of tagged proteins such asthat used in TAP (Tandem Affinity Purification) technology Rigaut et al.Nat Biotechnol. 1999 October; 17(10): 1030-2.

Preferably, BZR polypeptides useful in the methods of the invention haveDNA binding activity and bind a DNA fragment of preferably and inincreasing order of preference at least 20, 30, 40, 50, 75, 100, 150,200, 250, 300, nucleotides long comprising a BRRE element(Brassinosteroid response element) as represented by SEQ ID NO: 327element and/or an E-box element as represented by SEQ ID NO: 328. BRREelements and E-box elements are well known in the art (He et al. 2005;Yin et al 2008). Preferably the BRRE element and the E-box elementcomprises a sequence having at least 70%, 80%, 85%, 90%, 95% sequenceidentity to the sequence CGTGC(T/C)G (BRRE element: SEQ ID NO: 90) andCANNTC (E-box: SEQ ID NO: 328) respectively. More preferably the DNAfragment to which the BZR polypeptide binds is selected from the CPD,DWF4, UBC and CNX5 promoter as described by He et al. 2005.

In addition, BZR polypeptides, when expressed in rice according to themethods of the present invention as outlined in the Examples section,give plants having increased seed yield, in particular increased numberof filled seeds.

Concerning RHL1 polypeptides, the present invention is illustrated bytransforming plants with the nucleic acid sequence represented by SEQ IDNO: 1, encoding the polypeptide sequence of SEQ ID NO: 2. However,performance of the invention is not restricted to these sequences; themethods of the invention may advantageously be performed using anyRHL1-encoding nucleic acid or RHL1 polypeptide as defined herein.

Concerning RHL1 polypeptides, examples of nucleic acids encoding RHL1polypeptides are given in Table A1 of Example 1 herein. Such nucleicacids are useful in performing the methods of the invention. The aminoacid sequences given in Table A1 of Example 1 are example sequences oforthologues and paralogues of the RHL1 polypeptide represented by SEQ IDNO: 2, the terms “orthologues” and “paralogues” being as defined herein.Further orthologues and paralogues may readily be identified byperforming a so-called reciprocal blast search. Typically, this involvesa first BLAST involving BLASTing a query sequence (for example using anyof the sequences listed in Table A1 of Example 1) against any sequencedatabase, such as the publicly available NCBI database. BLASTN orTBLASTX (using standard default values) are generally used when startingfrom a nucleotide sequence, and BLASTP or TBLASTN (using standarddefault values) when starting from a protein sequence. The BLAST resultsmay optionally be filtered. The full-length sequences of either thefiltered results or non-filtered results are then BLASTed back (secondBLAST) against sequences from the organism from which the query sequenceis derived (where the query sequence is SEQ ID NO: 1 or SEQ ID NO: 2,the second BLAST would therefore be against Arabidopsis thalianasequences). The results of the first and second BLASTs are thencompared. A paralogue is identified if a high-ranking hit from the firstblast is from the same species as from which the query sequence isderived, a BLAST back then ideally results in the query sequence amongstthe highest hits; an orthologue is identified if a high-ranking hit inthe first BLAST is not from the same species as from which the querysequence is derived, and preferably results upon BLAST back in the querysequence being among the highest hits.

Concerning TGase polypeptides, the present invention is illustrated bytransforming plants with the nucleic acid sequence represented by SEQ IDNO: 44, encoding the TGase polypeptide sequence of SEQ ID NO: 45.However, performance of the invention is not restricted to thesesequences; the methods of the invention may advantageously be performedusing any nucleic acid sequence encoding a TGase polypeptide as definedherein.

Concerning TGase polypeptides, examples of nucleic acid sequencesencoding TGase polypeptides are given in Table A2 of Example 1 herein.Such nucleic acid sequences are useful in performing the methods of theinvention. The polypeptide sequences given in Table A2 of Example 1 areexample sequences of orthologues and paralogues of the TGase polypeptiderepresented by SEQ ID NO: 45, the terms “orthologues” and “paralogues”being as defined herein. Further orthologues and paralogues may readilybe identified by performing a so-called reciprocal blast search.Typically, this involves a first BLAST involving BLASTing a querysequence (for example using any of the sequences listed in Table A ofExample 1) against any sequence database, such as the publicly availableNCBI database. BLASTN or TBLASTX (using standard default values) aregenerally used when starting from a nucleotide sequence, and BLASTP orTBLASTN (using standard default values) when starting from a proteinsequence. The BLAST results may optionally be filtered. The full-lengthsequences of either the filtered results or non-filtered results arethen BLASTed back (second BLAST) against sequences from the organismfrom which the query sequence is derived (where the query sequence isSEQ ID NO: 44 or SEQ ID NO: 45, the second BLAST would therefore beagainst Oryza sativa sequences). The results of the first and secondBLASTs are then compared. A paralogue is identified if a high-rankinghit from the first blast is from the same species as from which thequery sequence is derived, a BLAST back then ideally results in thequery sequence amongst the highest hits; an orthologue is identified ifa high-ranking hit in the first BLAST is not from the same species asfrom which the query sequence is derived, and preferably results uponBLAST back in the query sequence being among the highest hits.

Concerning TRY-like polypeptides, the present invention is illustratedby transforming plants with the nucleic acid sequence represented by SEQID NO: 75, encoding the polypeptide sequence of SEQ ID NO: 76. However,performance of the invention is not restricted to these sequences; themethods of the invention may advantageously be performed using anyTRY-like-encoding nucleic acid or TRY-like polypeptide as definedherein.

Concerning TRY-like polypeptides, examples of nucleic acids encodingTRY-like polypeptides are given in Table A3 of Example 1 herein. Suchnucleic acids are useful in performing the methods of the invention. Theamino acid sequences given in Table A3 of Example 1 are examplesequences of orthologues and paralogues of the TRY-like polypeptiderepresented by SEQ ID NO: 76, the terms “orthologues” and “paralogues”being as defined herein. Further orthologues and paralogues may readilybe identified by performing a so-called reciprocal blast search.Typically, this involves a first BLAST involving BLASTing a querysequence (for example using any of the sequences listed in Table A3 ofExample 1) against any sequence database, such as the publicly availableNCBI database. BLASTN or TBLASTX (using standard default values) aregenerally used when starting from a nucleotide sequence, and BLASTP orTBLASTN (using standard default values) when starting from a proteinsequence. The BLAST results may optionally be filtered. The full-lengthsequences of either the filtered results or non-filtered results arethen BLASTed back (second BLAST) against sequences from the organismfrom which the query sequence is derived (where the query sequence isSEQ ID NO: 75 or SEQ ID NO: 76, the second BLAST would therefore beagainst Arabidopsis sequences). The results of the first and secondBLASTs are then compared. A paralogue is identified if a high-rankinghit from the first blast is from the same species as from which thequery sequence is derived, a BLAST back then ideally results in thequery sequence amongst the highest hits; an orthologue is identified ifa high-ranking hit in the first BLAST is not from the same species asfrom which the query sequence is derived, and preferably results uponBLAST back in the query sequence being among the highest hits.

Concerning BZR polypeptides, the present invention is illustrated bytransforming plants with the nucleic acid sequence represented by SEQ IDNO: 238, encoding the polypeptide sequence of SEQ ID NO: 239. However,performance of the invention is not restricted to these sequences; themethods of the invention may advantageously be performed using anyBZR-encoding nucleic acid or BZR polypeptide as defined herein.

Concerning BZR polypeptides, examples of nucleic acids encoding BZRpolypeptides are given in Table A4 of Example 1 herein. Such nucleicacids are useful in performing the methods of the invention. The aminoacid sequences given in Table A4 of Example 1 are example sequences oforthologues and paralogues of the BZR polypeptide represented by SEQ IDNO: 239, the terms “orthologues” and “paralogues” being as definedherein. Further orthologues and paralogues may readily be identified byperforming a so-called reciprocal blast search. Typically, this involvesa first BLAST involving BLASTing a query sequence (for example using anyof the sequences listed in Table A4 of Example 1) against any sequencedatabase, such as the publicly available NCBI database. BLASTN orTBLASTX (using standard default values) are generally used when startingfrom a nucleotide sequence, and BLASTP or TBLASTN (using standarddefault values) when starting from a protein sequence. The BLAST resultsmay optionally be filtered. The full-length sequences of either thefiltered results or non-filtered results are then BLASTed back (secondBLAST) against sequences from the organism from which the query sequenceis derived (where the query sequence is SEQ ID NO: 238 or SEQ ID NO:239, the second BLAST would therefore be against Arabidopsis thalianasequences). The results of the first and second BLASTs are thencompared. A paralogue is identified if a high-ranking hit from the firstblast is from the same species as from which the query sequence isderived, a BLAST back then ideally results in the query sequence amongstthe highest hits; an orthologue is identified if a high-ranking hit inthe first BLAST is not from the same species as from which the querysequence is derived, and preferably results upon BLAST back in the querysequence being among the highest hits.

High-ranking hits are those having a low E-value. The lower the E-value,the more significant the score (or in other words the lower the chancethat the hit was found by chance). Computation of the E-value is wellknown in the art. In addition to E-values, comparisons are also scoredby percentage identity. Percentage identity refers to the number ofidentical nucleotides (or amino acids) between the two compared nucleicacid (or polypeptide) sequences over a particular length. In the case oflarge families, ClustalW may be used, followed by a neighbour joiningtree, to help visualize clustering of related genes and to identifyorthologues and paralogues.

Concerning BZR polypeptides, preferably, the BZR polynucleotides usefulin the methods of the invention encode a polypeptide having inincreasing order of preference 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to the amino acid sequenceof any of the polypeptides of Table A4.

Nucleic acid variants may also be useful in practising the methods ofthe invention. Examples of such variants include nucleic acid sequencesencoding homologues and derivatives of any one of the amino acidsequences given in Table A1 to A4 of Example 1, the terms “homologue”and “derivative” being as defined herein. Also useful in the methods ofthe invention are nucleic acid sequences encoding homologues andderivatives of orthologues or paralogues of any one of the amino acidsequences given in Table A1 to A4 of Example 1. Homologues andderivatives useful in the methods of the present invention havesubstantially the same biological and functional activity as theunmodified protein from which they are derived. Further variants usefulin practising the methods of the invention are variants in which codonusage is optimised or in which miRNA target sites are removed.

Further nucleic acid variants useful in practising the methods of theinvention include portions of nucleic acid sequences encoding RHL1polypeptides, or TGase polypeptides, or TRY-like polypeptides, or BZRpolypeptides, nucleic acid sequences hybridising to nucleic acidsequences encoding RHL1 polypeptides, or TGase polypeptides, or TRY-likepolypeptides, or BZR polypeptides, splice variants of nucleic acidsequences encoding RHL1 polypeptides, or TRY-like polypeptides, or BZRpolypeptides, allelic variants of nucleic acids encoding RHL1polypeptides, or TGase polypeptides, or TRY-like polypeptides, or BZRpolypeptides, and variants of nucleic acid sequences encoding RHL1polypeptides, or TGase polypeptides, or TRY-like polypeptides, or BZRpolypeptides, obtained by gene shuffling. The terms hybridisingsequence, splice variant, allelic variant and gene shuffling are asdescribed herein.

Nucleic acids encoding RHL1 polypeptides, or TGase polypeptides, orTRY-like polypeptides, or BZR polypeptides, need not be full-lengthnucleic acids, since performance of the methods of the invention doesnot rely on the use of full-length nucleic acid sequences. According tothe present invention, there is provided a method for enhancingyield-related traits in plants, comprising introducing and expressing ina plant a portion of any one of the nucleic acid sequences given inTable A1 to A4 of Example 1, or a portion of a nucleic acid encoding anorthologue, paralogue or homologue of any of the amino acid sequencesgiven in Table A1 to A4 of Example 1.

A portion of a nucleic acid may be prepared, for example, by making oneor more deletions to the nucleic acid. The portions may be used inisolated form or they may be fused to other coding (or non-coding)sequences in order to, for example, produce a protein that combinesseveral activities. When fused to other coding sequences, the resultantpolypeptide produced upon translation may be bigger than that predictedfor the protein portion.

Concerning RHL1 polypeptides, portions useful in the methods of theinvention, encode a RHL1 polypeptide as defined herein, and havesubstantially the same biological activity as the amino acid sequencesgiven in Table A1 of Example 1. Preferably, the portion is a portion ofany one of the nucleic acids given in Table A1 of Example 1, or is aportion of a nucleic acid encoding an orthologue or paralogue of any oneof the amino acid sequences given in Table A1 of Example 1. Preferablythe portion is at least 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000 consecutive nucleotides in length, the consecutive nucleotidesbeing of any one of the nucleic acid sequences given in Table A1 ofExample 1, or of a nucleic acid encoding an orthologue or paralogue ofany one of the amino acid sequences given in Table A1 of Example 1. Mostpreferably the portion is a portion of the nucleic acid of SEQ ID NO: 1.Preferably, the portion encodes a fragment of an amino acid sequencewhich, when used in the construction of a phylogenetic tree, such as theone depicted in FIG. 3, clusters with any of the RHL1 polypeptidesoriginating from a dicotyledoneous plant comprising the amino acidsequence represented by SEQ ID NO: 2 rather than with any other group.

Concerning TGase polypeptides, portions useful in the methods of theinvention, encode a TGase polypeptide as defined herein, and havesubstantially the same biological activity as the polypeptide sequencesgiven in Table A2 of Example 1. Preferably, the portion is a portion ofany one of the nucleic acid sequences given in Table A2 of Example 1, oris a portion of a nucleic acid sequence encoding an orthologue orparalogue of any one of the polypeptide sequences given in Table A2 ofExample 1. Preferably the portion is, in increasing order of preferenceat least 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150,1200 or more consecutive nucleotides in length, the consecutivenucleotides being of any one of the nucleic acid sequences given inTable A2 of Example 1, or of a nucleic acid sequence encoding anorthologue or paralogue of any one of the polypeptide sequences given inTable A2 of Example 1. Preferably, the portion is a portion of a nucleicsequence encoding a polypeptide sequence having in increasing order ofpreference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, 99% or more amino acid sequence identity to the TGase polypeptideas represented by SEQ ID NO: 45 or to any of the polypeptide sequencesgiven in Table A herein. Most preferably, the portion is a portion ofthe nucleic acid sequence of SEQ ID NO: 44.

Concerning TRY-like polypeptides, portions useful in the methods of theinvention, encode a TRY-like polypeptide as defined herein, and havesubstantially the same biological activity as the amino acid sequencesgiven in Table A3 of the Example 1. Preferably, the portion is a portionof any one of the nucleic acids given in Table A3 of the Example 1, oris a portion of a nucleic acid encoding an orthologue or paralogue ofany one of the amino acid sequences given in Table A3 of the Example 1.Preferably the portion is at least 150, 200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700,1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300,2350, 2400, 2450, 2500 consecutive nucleotides in length, theconsecutive nucleotides being of any one of the nucleic acid sequencesgiven in Table A3 of the Example 1, or of a nucleic acid encoding anorthologue or paralogue of any one of the amino acid sequences given inTable A3 of the Example 1. Most preferably the portion is a portion ofthe nucleic acid of SEQ ID NO: 75. Preferably, the portion encodes afragment of an polypeptide comprising a Myb-like DNA-binding domain(PFam domain PF00249.17, SMART domain SM00717, ProfileScan domainPS50090, Panther PTHR10641:SF26).

Concerning BZR polypeptides, portions useful in the methods of theinvention, encode a BZR polypeptide as defined herein, and havesubstantially the same biological activity as the amino acid sequencesgiven in Table A4 of The Example 1. Preferably, the portion is a portionof any one of the nucleic acids given in Table A4 of The Example 1, oris a portion of a nucleic acid encoding an orthologue or paralogue ofany one of the amino acid sequences given in Table A4 of The Example 1.Preferably the portion is at least 20, 25, 30, 35, 40, 45, 50, 75, 100,150, 200, 250, 300, 350, 400, 500, 550, 600, 700, 800, 900 consecutivenucleotides in length, the consecutive nucleotides being of any one ofthe nucleic acid sequences given in Table A4 of The Example 1, or of anucleic acid encoding an orthologue or paralogue of any one of the aminoacid sequences given Table A4 of The Example 1. Most preferably theportion is a portion of the nucleic acid of SEQ ID NO: 238. Preferably,the portion encodes a fragment of an amino acid sequence comprising aprotein domain having in increasing order of preference 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to a bHLH-like domain asrepresented SEQ ID NO: 326.

Another nucleic acid variant useful in the methods of the invention is anucleic acid sequence capable of hybridising, under reduced stringencyconditions, preferably under stringent conditions, with a nucleic acidencoding a RHL1 polypeptide, or a TGase polypeptide, or a TRY-likepolypeptide, or a BZR polypeptide, as defined herein, or with a portionas defined herein.

Concerning RHL1 polypeptides, or TRY-like polypeptides, according to thepresent invention, there is provided a method for enhancingyield-related traits in plants, comprising introducing and expressing ina plant a nucleic acid capable of hybridizing to any one of the nucleicacids given in Table A1, or Table A3 of Example 1, or comprisingintroducing and expressing in a plant a nucleic acid capable ofhybridising to a nucleic acid encoding an orthologue, paralogue orhomologue of any of the nucleic acid sequences given in Table A1, orTable A3 of Example 1.

Concerning TGase polypeptides, according to the present invention, thereis provided a method for increasing seed yield-related traits in plants,comprising introducing and expressing in a plant, a nucleic acidsequence capable of hybridizing to any one of the nucleic acid sequencesgiven in Table A2 of Example 1, or comprising introducing and expressingin a plant, a nucleic acid sequence capable of hybridising to a nucleicacid sequence encoding an orthologue, paralogue or homologue of any ofthe nucleic acid sequences given in Table A2 of Example 1.

Concerning BZR polypeptides, according to the present invention, thereis provided a method for increasing seed yield in plants, comprisingintroducing and expressing in a plant a nucleic acid capable ofhybridizing to any one of the nucleic acids given in Table A4 of Example1, or comprising introducing and expressing in a plant a nucleic acidcapable of hybridising to a nucleic acid encoding an orthologue,paralogue or homologue of any of the nucleic acid sequences given inTable A4 of Example 1.

Concerning RHL1 polypeptides, hybridising sequences useful in themethods of the invention encode a RHL1 polypeptide as defined herein,having substantially the same biological activity as the amino acidsequences given in Table A1 of Example 1. Preferably, the hybridisingsequence is capable of hybridising to any one of the nucleic acids givenin Table A1 of Example 1, or to a portion of any of these sequences, aportion being as defined above, or the hybridising sequence is capableof hybridising to a nucleic acid encoding an orthologue or paralogue ofany one of the amino acid sequences given in Table A1 of Example 1. Mostpreferably, the hybridising sequence is capable of hybridising to anucleic acid as represented by SEQ ID NO: 1 or to a portion thereof.

Concerning TGase polypeptides, hybridising sequences useful in themethods of the invention encode a TGase polypeptide as defined herein,and have substantially the same biological activity as the polypeptidesequences given in Table A2 of Example 1. Preferably, the hybridisingsequence is capable of hybridising to any one of the nucleic acidsequences given in Table A2 of Example 1, or to a complement thereof, orto a portion of any of these sequences, a portion being as definedabove, or wherein the hybridising sequence is capable of hybridising toa nucleic acid sequence encoding an orthologue or paralogue of any oneof the polypeptide sequences given in Table A2 of Example 1, or to acomplement thereof. Preferably, the hybridising sequence is capable ofhybridising to a nucleic acid sequence encoding a polypeptide sequencehaving in increasing order of preference at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequenceidentity to the TGase polypeptide as represented by SEQ ID NO: 45 or toany of the polypeptide sequences given in Table A herein. Mostpreferably, the hybridising sequence is capable of hybridising to anucleic acid sequence as represented by SEQ ID NO: 44 or to a portionthereof.

Concerning TRY-like polypeptides, hybridising sequences useful in themethods of the invention encode a TRY-like polypeptide as definedherein, having substantially the same biological activity as the aminoacid sequences given in Table A3 of Example 1. Preferably, thehybridising sequence is capable of hybridising to the complement of anyone of the nucleic acids given in Table A3 of Example 1, or to a portionof any of these sequences, a portion being as defined above, or thehybridising sequence is capable of hybridising to the complement of anucleic acid encoding an orthologue or paralogue of any one of the aminoacid sequences given in Table A3 of Example 1. Most preferably, thehybridising sequence is capable of hybridising to the complement of anucleic acid as represented by SEQ ID NO: 75 or to a portion thereof.

Concerning BZR polypeptides, hybridising sequences useful in the methodsof the invention encode a BZR polypeptide as defined herein, havingsubstantially the same biological activity as the amino acid sequencesgiven in Table A4 of Example 1. Preferably, the hybridising sequence iscapable of hybridising to the complement of any one of the nucleic acidsgiven in Table A4 of Example 1, or to a portion of any of thesesequences, a portion being as defined above, or the hybridising sequenceis capable of hybridising to the complement of a nucleic acid encodingan orthologue or paralogue of any one of the amino acid sequences givenin Table A4 of Example 1. Most preferably, the hybridising sequence iscapable of hybridising to the complement of a nucleic acid asrepresented by SEQ ID NO: 238 or to a portion thereof.

Concerning RHL1 polypeptides, preferably, the hybridising sequenceencodes a polypeptide with an amino acid sequence which, whenfull-length and used in the construction of a phylogenetic tree, such asthe one depicted in FIG. 3, clusters with any of the RHL1 polypeptidesoriginating from a dicotyledoneous plant comprising the amino acidsequence represented by SEQ ID NO: 2 rather than with any other group.

Concerning TRY-like polypeptides, preferably, the hybridising sequenceencodes a polypeptide comprising a Myb-like DNA-binding domain (PFamdomain PF00249.17, SMART domain SM00717, ProfileScan domain PS50090,Panther PTHR10641:SF26).

Concerning BZR polypeptides, preferably, the hybridising sequencecomprises a protein domain or encodes a polypeptide with an amino acidsequence which, when full-length comprises a protein domain having inincreasing order of preference 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to a bHLH-like domain as represented SEQ ID NO: 326.

Another nucleic acid sequence variant useful in the methods of theinvention is a splice variant encoding a RHL1 polypeptide, or a TGasepolypeptide, or a TRY-like polypeptide, or a BZR polypeptide, as definedhereinabove, a splice variant being as defined herein.

Concerning RHL1 polypeptides, or TRY-like polypeptides, according to thepresent invention, there is provided a method for enhancingyield-related traits in plants, comprising introducing and expressing ina plant a splice variant of any one of the nucleic acid sequences givenin Table A1, or Table A3 of Example 1, or a splice variant of a nucleicacid encoding an orthologue, paralogue or homologue of any of the aminoacid sequences given in Table A1, or Table A3 of Example 1.

Concerning TGase polypeptides, according to the present invention, thereis provided a method for increasing seed yield-related traits,comprising introducing and expressing in a plant, a splice variant ofany one of the nucleic acid sequences given in Table A2 of Example 1, ora splice variant of a nucleic acid sequence encoding an orthologue,paralogue or homologue of any of the polypeptide sequences given inTable A2 of Example 1, having substantially the same biological activityas the polypeptide sequence as represented by SEQ ID NO: 45 and any ofthe polypeptide sequences depicted in Table A2 of Example 1.

Concerning BZR polypeptides, according to the present invention, thereis provided a method for increasing seed yield in plants, comprisingintroducing and expressing in a plant a splice variant of any one of thenucleic acid sequences given in Table A4 of Example 1, or a splicevariant of a nucleic acid encoding an orthologue, paralogue or homologueof any of the amino acid sequences given in Table A4 of Example 1.

Concerning RHL1 polypeptides, preferred splice variants are splicevariants of a nucleic acid represented by SEQ ID NO: 1, or a splicevariant of a nucleic acid encoding an orthologue or paralogue of SEQ IDNO: 2. Preferably, the amino acid sequence encoded by the splicevariant, when used in the construction of a phylogenetic tree, such asthe one depicted in FIG. 3, clusters with any of the RHL1 polypeptidesoriginating from a dicotyledoneous plant comprising the amino acidsequence represented by SEQ ID NO: 2 rather than with any other group.

Concerning TGase polypeptides, preferred splice variants are splicevariants of a nucleic acid sequence represented by SEQ ID NO: 44, or asplice variant of a nucleic acid sequence encoding an orthologue orparalogue of SEQ ID NO: 45. Preferably, the splice variant is a splicevariant of a nucleic acid sequence encoding a polypeptide sequencehaving in increasing order of preference at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequenceidentity to the TGase polypeptide as represented by SEQ ID NO: 45 or toany of the polypeptide sequences given in Table A2 herein.

Concerning TRY-like polypeptides, preferred splice variants are splicevariants of a nucleic acid represented by SEQ ID NO: 75, or a splicevariant of a nucleic acid encoding an orthologue or paralogue of SEQ IDNO: 76. Preferably, the amino acid sequence encoded by the splicevariant comprises a Myb-like DNA-binding domain (PFam domain PF00249.17,SMART domain SM00717, ProfileScan domain PS50090, PantherPTHR10641:SF26).

Concerning BZR polypeptides, preferred splice variants are splicevariants of a nucleic acid represented by SEQ ID NO: 238, or a splicevariant of a nucleic acid encoding an orthologue or paralogue of SEQ IDNO: 239. Preferably, the amino acid sequence encoded by the splicevariant comprises a protein domain having in increasing order ofpreference 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity toa bHLH-like domain as represented SEQ ID NO: 326.

Another nucleic acid sequence variant useful in performing the methodsof the invention is an allelic variant of a nucleic acid encoding a RHL1polypeptide, or a TGase polypeptide, or TRY-like polypeptide, or a BZRpolypeptide, as defined hereinabove, an allelic variant being as definedherein.

Concerning RHL1 polypeptides, or TRY-like polypeptides, according to thepresent invention, there is provided a method for enhancingyield-related traits in plants, comprising introducing and expressing ina plant an allelic variant of any one of the nucleic acids given inTable A1, or Table A3 of Example 1, or comprising introducing andexpressing in a plant an allelic variant of a nucleic acid encoding anorthologue, paralogue or homologue of any of the amino acid sequencesgiven in Table A1, or Table A3 of Example 1.

Concerning TGase polypeptides, according to the present invention, thereis provided a method for increasing seed yield-related traits,comprising introducing and expressing in a plant, an allelic variant ofany one of the nucleic acid sequences given in Table A2 of Example 1, orcomprising introducing and expressing in a plant, an allelic variant ofa nucleic acid sequence encoding an orthologue, paralogue or homologueof any of the polypeptide sequences given in Table A2 of Example 1.

Concerning BZR polypeptides, according to the present invention, thereis provided a method for increasing seed yield in plants, comprisingintroducing and expressing in a plant an allelic variant of any one ofthe nucleic acids given in Table A4 of Example 1, or comprisingintroducing and expressing in a plant an allelic variant of a nucleicacid encoding an orthologue, paralogue or homologue of any of the aminoacid sequences given in Table A4 of Example 1.

Concerning RHL1 polypeptides, the polypeptides encoded by allelicvariants useful in the methods of the present invention havesubstantially the same biological activity as the RHL1 polypeptide ofSEQ ID NO: 2 and any of the amino acids depicted in Table A1 ofExample 1. Allelic variants exist in nature, and encompassed within themethods of the present invention is the use of these natural alleles.Preferably, the allelic variant is an allelic variant of SEQ ID NO: 1 oran allelic variant of a nucleic acid encoding an orthologue or paralogueof SEQ ID NO: 2. Preferably, the amino acid sequence encoded by theallelic variant, when used in the construction of a phylogenetic tree,such as the one depicted in FIG. 3, clusters with any of the RHL1polypeptides originating from a dicotyledoneous plant comprising theamino acid sequence represented by SEQ ID NO: 2 rather than with anyother group.

Concerning TGase polypeptides, the allelic variants useful in themethods of the present invention have substantially the same biologicalactivity as the TGase polypeptide of SEQ ID NO: 45 and any of thepolypeptide sequences depicted in Table A2 of Example 1. Allelicvariants exist in nature, and encompassed within the methods of thepresent invention is the use of these natural alleles. Preferably, theallelic variant is an allelic variant of SEQ ID NO: 44 or an allelicvariant of a nucleic acid sequence encoding an orthologue or paralogueof SEQ ID NO: 45. Preferably, the allelic variant is an allelic variantof a polypeptide sequence having in increasing order of preference atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or moreamino acid sequence identity to the TGase polypeptide as represented bySEQ ID NO: 45 or to any of the polypeptide sequences given in Table A2herein.

Concerning TRY-like polypeptides, the polypeptides encoded by allelicvariants useful in the methods of the present invention havesubstantially the same biological activity as the TRY-like polypeptideof SEQ ID NO: 76 and any of the amino acids depicted in Table A3 ofExample 1. Allelic variants exist in nature, and encompassed within themethods of the present invention is the use of these natural alleles.Allelic variants of SEQ ID NO: 76 are for example described inSchellmann et al. (2002). Preferably, the allelic variant is an allelicvariant of SEQ ID NO: 75 or an allelic variant of a nucleic acidencoding an orthologue or paralogue of SEQ ID NO: 76. Preferably, theamino acid sequence encoded by the allelic variant, polypeptidecomprises a Myb-like DNA-binding domain (PFam domain PF00249.17, SMARTdomain SM00717, ProfileScan domain PS50090, Panther PTHR10641:SF26).

Concerning BZR polypeptides, the polypeptides encoded by allelicvariants useful in the methods of the present invention havesubstantially the same biological activity as the BZR polypeptide of SEQID NO: 239 and any of the amino acids depicted in Table A4 of Example 1.Allelic variants exist in nature, and encompassed within the methods ofthe present invention is the use of these natural alleles. Preferably,the allelic variant is an allelic variant of SEQ ID NO: 238 or anallelic variant of a nucleic acid encoding an orthologue or paralogue ofSEQ ID NO: 239. Preferably, the amino acid sequence encoded by theallelic comprises a protein domain having in increasing order ofpreference 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity toa bHLH-like domain as represented SEQ ID NO: 326.

Gene shuffling or directed evolution may also be used to generatevariants of nucleic acid sequences encoding RHL1 polypeptides, or TGasepolypeptides, or TRY-like polypeptides, or BZR polypeptides, as definedabove; the term “gene shuffling” being as defined herein.

Concerning RHL1 polypeptides, or TRY-like polypeptides, according to thepresent invention, there is provided a method for enhancingyield-related traits in plants, comprising introducing and expressing ina plant a variant of any one of the nucleic acid sequences given inTable A1, or Table A3 of Example 1, or comprising introducing andexpressing in a plant a variant of a nucleic acid encoding anorthologue, paralogue or homologue of any of the amino acid sequencesgiven in Table A1, or Table A3 of Example 1, which variant nucleic acidis obtained by gene shuffling.

Concerning TGase polypeptides, according to the present invention, thereis provided a method for increasing seed yield-related traits,comprising introducing and expressing in a plant, a variant of any oneof the nucleic acid sequences given in Table A2 of Example 1, orcomprising introducing and expressing in a plant a variant of a nucleicacid sequence encoding an orthologue, paralogue or homologue of any ofthe polypeptide sequences given in Table A2 of Example 1, which variantnucleic acid sequence is obtained by gene shuffling.

Concerning BZR polypeptides, according to the present invention, thereis provided a method for increasing seed yield in plants, comprisingintroducing and expressing in a plant a variant of any one of thenucleic acid sequences given in Table A4 of Example 1, or comprisingintroducing and expressing in a plant a variant of a nucleic acidencoding an orthologue, paralogue or homologue of any of the amino acidsequences given in Table A4 of Example 1, which variant nucleic acid isobtained by gene shuffling.

Concerning RHL1 polypeptides, preferably, the amino acid sequenceencoded by the variant nucleic acid obtained by gene shuffling, whenused in the construction of a phylogenetic tree such as the one depictedin FIG. 3, clusters with any of the RHL1 polypeptides originating from adicotyledoneous plant comprising the amino acid sequence represented bySEQ ID NO: 2 rather than with any other group.

Concerning TGase polypeptides, preferably, the variant nucleic acidsequence obtained by gene shuffling encodes a polypeptide sequencehaving in increasing order of preference at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequenceidentity to the TGase polypeptide as represented by SEQ ID NO: 45 or toany of the polypeptide sequences given in Table A2 herein.

Concerning TRY-like polypeptides, preferably, the amino acid sequenceencoded by the variant nucleic acid obtained by gene shuffling,comprises a Myb-like DNA-binding domain (PFam domain PF00249.17, SMARTdomain SM00717, ProfileScan domain PS50090, Panther PTHR10641:SF26).

Concerning BZR polypeptides, preferably, the amino acid sequence encodedby the variant nucleic acid obtained by gene shuffling comprises aprotein domain having in increasing order of preference 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to a bHLH-like domain asrepresented SEQ ID NO: 326.

Furthermore, nucleic acid variants may also be obtained by site-directedmutagenesis. Several methods are available to achieve site-directedmutagenesis, the most common being PCR based methods (Current Protocolsin Molecular Biology. Wiley Eds.).

Nucleic acids encoding RHL1 polypeptides may be derived from any naturalor artificial source. The nucleic acid may be modified from its nativeform in composition and/or genomic environment through deliberate humanmanipulation. Preferably the RHL1 polypeptide-encoding nucleic acid isfrom a plant, further preferably from a dicocotyledonous plant, morepreferably from the family Breassicaceae, most preferably the nucleicacid is from Arabidopsis thaliana.

Nucleic acid sequences encoding TGase polypeptides may be derived fromany natural or artificial source. The nucleic acid sequence may bemodified from its native form in composition and/or genomic environmentthrough deliberate human manipulation. The nucleic acid sequenceencoding a TGase polypeptide is from a plant, further preferably from adicotyledonous plant, more preferably from the family Poaceae, mostpreferably the nucleic acid sequence is from Oryza sativa.

Nucleic acids encoding TRY-like polypeptides may be derived from anynatural or artificial source. The nucleic acid may be modified from itsnative form in composition and/or genomic environment through deliberatehuman manipulation. Preferably the TRY-like polypeptide-encoding nucleicacid is from a plant, further preferably from a dicotyledonous plant,more preferably from the family Brassicaceae, most preferably thenucleic acid is from Arabidopsis thaliana.

Nucleic acids encoding BZR polypeptides may be derived from any naturalor artificial source. The nucleic acid may be modified from its nativeform in composition and/or genomic environment through deliberate humanmanipulation. Preferably the BZR polypeptide-encoding nucleic acid isfrom a plant, further preferably from a heterologous plant, morepreferably from a dicotyledonous plant, even more preferably from thefamily Brassicaceae, most preferably the nucleic acid is fromArabidopsis thaliana.

Advantageously, the present invention provides hitherto unknown BZRnucleic acid and polypeptide sequences.

According to a further embodiment of the present invention, there isprovided an isolated nucleic acid molecule comprising:

-   -   (i) a nucleic acid represented by any one of SEQ ID NO: 13, 15,        17, 19;    -   (ii) a nucleic acid or fragment thereof that is complementary to        any one of SEQ ID NO: 13, 15, 17, 19;    -   (iii) a nucleic acid encoding an BZR polypeptide having, in        increasing order of preference, at least 80%, 81%, 82%, 83%,        84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, or 99% or more sequence identity to one of SEQ ID NO:        14, 16, 18, 20;    -   (iv) a nucleic acid capable of hybridizing under stringent        conditions to any one of the nucleic acids given in (i), (ii)        or (iii) above.

According to a further embodiment of the present invention, there istherefore provided an isolated polypeptide comprising:

-   -   (i) an amino acid sequence having, in increasing order of        preference, at least at least 80%, 81%, 82%, 83%, 84%, 85%, 86%,        87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or        99% or more sequence identity to one of SEQ ID NO: 14, 16, 18,        20;    -   (ii) derivatives of any of the amino acid sequences given in        (i).

Concerning RHL1 polypeptides, performance of the methods of theinvention gives plants having enhanced yield-related traits. Inparticular performance of the methods of the invention gives plantshaving increased yield, especially increased seed yield relative tocontrol plants. The terms “yield” and “seed yield” are described in moredetail in the “definitions” section herein.

Concerning TGase polypeptides, performance of the methods of theinvention gives plants having increased seed yield-related traitsrelative to control plants. The terms “yield” and “seed yield” aredescribed in more detail in the “definitions” section herein.

Concerning TRY-like polypeptides, performance of the methods of theinvention gives plants having enhanced yield-related traits. Inparticular performance of the methods of the invention gives plantshaving increased early vigour (emergence vigour) and increased yield,especially increased seed yield relative to control plants. The terms“early vigour”, “yield” and “seed yield” are described in more detail inthe “definitions” section herein.

Concerning BZR polypeptides, performance of the methods of the inventiongives plants having increased seed yield

Concerning RHL1 polypeptides, reference herein to enhanced yield-relatedtraits is taken to mean an increase in biomass (weight) of one or moreparts of a plant, which may include aboveground (harvestable) partsand/or (harvestable) parts below ground. In particular, such harvestableparts are seeds, and performance of the methods of the invention resultsin plants having increased seed yield relative to the seed yield ofcontrol plants.

Concerning TRY-like polypeptides, reference herein to enhancedyield-related traits is taken to mean an increase in early vigour and/oran increase in biomass (weight) of one or more parts of a plant, whichmay include aboveground (harvestable) parts and/or (harvestable) partsbelow ground. In particular, such harvestable parts are seeds, andperformance of the methods of the invention results in plants havingincreased seed yield relative to the seed yield of control plants.

Concerning BZR polypeptides, reference herein to increase seed yield istaken to mean any one or more of the following seed parameters: anincrease in the seed weight, the total number of seed, the number offilled seeds, the seed filing rate, the proportion of filled seeds, thesize of the seed, the volume of the seed harvested. The skill in artwill recognized that the abovementioned seed yield parameters may beexpressed in different units including but not limited to per panicleand/or per plant and/or per harvest. Performance of the methods of theinvention results in plants having increased seed yield relative to theseed yield of control plants.

Taking corn as an example, a yield increase may be manifested as one ormore of the following: increase in the number of plants established persquare meter, an increase in the number of ears per plant, an increasein the number of rows, number of kernels per row, kernel weight,thousand kernel weight, ear length/diameter, increase in the seedfilling rate (which is the number of filled seeds divided by the totalnumber of seeds and multiplied by 100), among others. Taking rice as anexample, a yield increase may manifest itself as an increase in one ormore of the following: number of plants per square meter, number ofpanicles per plant, number of spikelets per panicle, number of flowers(florets) per panicle (which is expressed as a ratio of the number offilled seeds over the number of primary panicles), increase in the seedfilling rate (which is the number of filled seeds divided by the totalnumber of seeds and multiplied by 100), increase in thousand kernelweight, among others.

The present invention provides a method for increasing yield, especiallyseed yield of plants, relative to control plants, which method comprisesmodulating expression in a plant of a nucleic acid encoding a RHL1polypeptide, or a TRY-like polypeptide, as defined herein.

The present invention also provides a method for increasing seedyield-related traits of plants relative to control plants, which methodcomprises increasing expression in a plant of a nucleic acid sequenceencoding a TGase polypeptide as defined herein.

The present invention furthermore provides a method for increasing seedyield of plants, relative to control plants, which method comprisesmodulating expression in a plant of a nucleic acid encoding a BZRpolypeptide as defined herein.

Since the transgenic plants according to the present invention haveincreased yield and/or increased seed yield-related traits and/or yield,it is likely that these plants exhibit an increased growth rate (duringat least part of their life cycle), relative to the growth rate ofcontrol plants at a corresponding stage in their life cycle.

The increased growth rate may be specific to one or more parts of aplant (including seeds), or may be throughout substantially the wholeplant. Plants having an increased growth rate may have a shorter lifecycle. The life cycle of a plant may be taken to mean the time needed togrow from a dry mature seed up to the stage where the plant has produceddry mature seeds, similar to the starting material. This life cycle maybe influenced by factors such as early vigour, growth rate, greennessindex, flowering time and speed of seed maturation. The increase ingrowth rate may take place at one or more stages in the life cycle of aplant or during substantially the whole plant life cycle. Increasedgrowth rate during the early stages in the life cycle of a plant mayreflect enhanced vigour. The increase in growth rate may alter theharvest cycle of a plant allowing plants to be sown later and/orharvested sooner than would otherwise be possible (a similar effect maybe obtained with earlier flowering time). If the growth rate issufficiently increased, it may allow for the further sowing of seeds ofthe same plant species (for example sowing and harvesting of rice plantsfollowed by sowing and harvesting of further rice plants all within oneconventional growing period). Similarly, if the growth rate issufficiently increased, it may allow for the further sowing of seeds ofdifferent plants species (for example the sowing and harvesting of cornplants followed by, for example, the sowing and optional harvesting ofsoybean, potato or any other suitable plant). Harvesting additionaltimes from the same rootstock in the case of some crop plants may alsobe possible. Altering the harvest cycle of a plant may lead to anincrease in annual biomass production per square meter (due to anincrease in the number of times (say in a year) that any particularplant may be grown and harvested). An increase in growth rate may alsoallow for the cultivation of transgenic plants in a wider geographicalarea than their wild-type counterparts, since the territoriallimitations for growing a crop are often determined by adverseenvironmental conditions either at the time of planting (early season)or at the time of harvesting (late season). Such adverse conditions maybe avoided if the harvest cycle is shortened. The growth rate may bedetermined by deriving various parameters from growth curves, suchparameters may be: T-Mid (the time taken for plants to reach 50% oftheir maximal size) and T-90 (time taken for plants to reach 90% oftheir maximal size), amongst others.

According to a preferred feature of the present invention, performanceof the methods of the invention gives plants having an increased growthrate relative to control plants. Therefore, according to the presentinvention, there is provided a method for increasing the growth rate ofplants, which method comprises modulating expression in a plant of anucleic acid sequence encoding a RHL1 polypeptide, or a TGasepolypeptide, or TRY-like polypeptide, a BZR polypeptide, as definedherein.

Increased seed yield-related traits occur whether the plant is undernon-stress conditions or whether the plant is exposed to variousstresses compared to control plants grown under comparable conditions.Plants typically respond to exposure to stress by growing more slowly.In conditions of severe stress, the plant may even stop growingaltogether. Mild stress on the other hand is defined herein as being anystress to which a plant is exposed which does not result in the plantceasing to grow altogether without the capacity to resume growth. Mildstress in the sense of the invention leads to a reduction in the growthof the stressed plants of less than 40%, 35% or 30%, preferably lessthan 25%, 20% or 15%, more preferably less than 14%, 13%, 12%, 11% or10% or less in comparison to the control plant under non-stressconditions. Due to advances in agricultural practices (irrigation,fertilization, pesticide treatments) severe stresses are not oftenencountered in cultivated crop plants. As a consequence, the compromisedgrowth induced by mild stress is often an undesirable feature foragriculture. Mild stresses are the everyday biotic and/or abiotic(environmental) stresses to which a plant is exposed. Abiotic stressesmay be due to drought or excess water, anaerobic stress, salt stress,chemical toxicity, oxidative stress and hot, cold or freezingtemperatures. The abiotic stress may be an osmotic stress caused by awater stress (particularly due to drought), salt stress, oxidativestress or an ionic stress. Biotic stresses are typically those stressescaused by pathogens, such as bacteria, viruses, fungi, nematodes, andinsects. The term “non-stress” conditions as used herein are thoseenvironmental conditions that allow optimal growth of plants. Personsskilled in the art are aware of normal soil conditions and climaticconditions for a given location.

In particular, the methods of the present invention may be performedunder non-stress conditions or under conditions of mild drought to giveplants having increased yield relative to control plants. As reported inWang et al. (Planta (2003) 218: 1-14), abiotic stress leads to a seriesof morphological, physiological, biochemical and molecular changes thatadversely affect plant growth and productivity. Drought, salinity,extreme temperatures and oxidative stress are known to be interconnectedand may induce growth and cellular damage through similar mechanisms.Rabbani et al. (Plant Physiol (2003) 133: 1755-1767) describes aparticularly high degree of “cross talk” between drought stress andhigh-salinity stress. For example, drought and/or salinisation aremanifested primarily as osmotic stress, resulting in the disruption ofhomeostasis and ion distribution in the cell. Oxidative stress, whichfrequently accompanies high or low temperature, salinity or droughtstress, may cause denaturing of functional and structural proteins. As aconsequence, these diverse environmental stresses often activate similarcell signalling pathways and cellular responses, such as the productionof stress proteins, up-regulation of anti-oxidants, accumulation ofcompatible solutes and growth arrest. The term “non-stress” conditionsas used herein are those environmental conditions that allow optimalgrowth of plants. Persons skilled in the art are aware of normal soilconditions and climatic conditions for a given location. Plants withoptimal growth conditions, (grown under non-stress conditions) typicallyyield in increasing order of preference at least 97%, 95%, 92%, 90%,87%, 85%, 83%, 80%, 77% or 75% of the average production of such plantin a given environment. Average production may be calculated on harvestand/or season basis. Persons skilled in the art are aware of averageyield productions of a crop.

The term “abiotic stress” as defined herein is taken to mean any one ormore of: water stress (due to drought or excess water), anaerobicstress, salt stress, temperature stress (due to hot, cold or freezingtemperatures), chemical toxicity stress and oxidative stress. Accordingto one aspect of the invention, the abiotic stress is an osmotic stress,selected from water stress, salt stress, oxidative stress and ionicstress. Preferably, the water stress is drought stress. The term saltstress is not restricted to common salt (NaCl), but may be any stresscaused by one or more of: NaCl, KCl, LiCl, MgCl₂, CaCl₂, amongst others.

Performance of the methods of the invention gives plants havingincreased seed yield-related traits, under abiotic stress conditionsrelative to control plants grown in comparable stress conditions.Therefore, according to the present invention, there is provided amethod for increasing seed yield-related traits, in plants grown underabiotic stress conditions, which method comprises increasing expressionin a plant of a nucleic acid sequence encoding a TGase polypeptide.According to one aspect of the invention, the abiotic stress is anosmotic stress, selected from one or more of the following: waterstress, salt stress, oxidative stress and ionic stress.

Another example of abiotic environmental stress is the reducedavailability of one or more nutrients that need to be assimilated by theplants for growth and development. Because of the strong influence ofnutrition utilization efficiency on plant yield and product quality, ahuge amount of fertilizer is poured onto fields to optimize plant growthand quality. Productivity of plants ordinarily is limited by threeprimary nutrients, phosphorous, potassium and nitrogen, which is usuallythe rate-limiting element in plant growth of these three. Therefore themajor nutritional element required for plant growth is nitrogen (N). Itis a constituent of numerous important compounds found in living cells,including amino acids, proteins (enzymes), nucleic acids, andchlorophyll. 1.5% to 2% of plant dry matter is nitrogen andapproximately 16% of total plant protein. Thus, nitrogen availability isa major limiting factor for crop plant growth and production (Frink etal. (1999) Proc Natl Acad Sci USA 96(4): 1175-1180), and has as well amajor impact on protein accumulation and amino acid composition.Therefore, of great interest are crop plants with increased seedyield-related traits, when grown under nitrogen-limiting conditions.

Performance of the methods of the invention gives plants grown underconditions of reduced nutrient availability, particularly underconditions of reduced nitrogen availablity, having increased seedyield-related traits relative to control plants grown under comparableconditions. Therefore, according to the present invention, there isprovided a method for increasing seed yield-related traits in plantsgrown under conditions of reduced nutrient availablity, preferablyreduced nitrogen availability, which method comprises increasingexpression in a plant of a nucleic acid sequence encoding a TGasepolypeptide. Reduced nutrient availability may result from a deficiencyor excess of nutrients such as nitrogen, phosphates and otherphosphorous-containing compounds, potassium, calcium, cadmium,magnesium, manganese, iron and boron, amongst others. Preferably,reduced nutrient availablity is reduced nitrogen availability.

Concerning RHL1 polypeptides, performance of the methods of theinvention gives plants grown under non-stress conditions or under milddrought conditions increased yield relative to control plants grownunder comparable conditions. Therefore, according to the presentinvention, there is provided a method for increasing yield in plantsgrown under non-stress conditions or under mild drought conditions,which method comprises modulating expression in a plant of a nucleicacid sequence encoding a RHL1 polypeptide.

Concerning TGase polypeptides, performance of the methods of theinvention gives plants grown under non-stress conditions or under mildstress conditions having increased seed yield-related traits, relativeto control plants grown under comparable conditions. Therefore,according to the present invention, there is provided a method forincreasing seed yield-related traits in plants grown under non-stressconditions or under mild stress conditions, which method comprisesincreasing expression in a plant of a nucleic acid sequence encoding aTGase polypeptide.

Concerning TRY-like polypeptides, performance of the methods of theinvention gives plants grown under non-stress conditions or under milddrought conditions increased yield relative to control plants grownunder comparable conditions. Therefore, according to one embodiment ofthe present invention, there is provided a method for increasing yieldin plants grown under non-stress conditions or under mild droughtconditions, which method comprises modulating expression in a plant of anucleic acid encoding a TRY-like polypeptide.

Concerning BZR polypeptides, performance of the methods of the inventiongives plants grown under non-stress conditions or under mild droughtconditions increased seed yield relative to control plants grown undercomparable conditions. Therefore, according to the present invention,there is provided a method for increasing seed yield in plants grownunder non-stress conditions or under mild drought conditions, whichmethod comprises modulating expression in a plant of a nucleic acidencoding a BZR polypeptide.

Concerning RHL1 polypeptides, performance of the methods of theinvention gives plants grown under conditions of nutrient deficiency,particularly under conditions of nitrogen deficiency, increased yieldrelative to control plants grown under comparable conditions. Therefore,according to the present invention, there is provided a method forincreasing yield in plants grown under conditions of nutrientdeficiency, which method comprises modulating expression in a plant of anucleic acid encoding a RHL1 polypeptide. Nutrient deficiency may resultfrom a lack of nutrients such as nitrogen, phosphates and otherphosphorous-containing compounds, potassium, calcium, cadmium,magnesium, manganese, iron and boron, amongst others.

Concerning TRY-like polypeptides, performance of the methods of theinvention gives plants grown under conditions of nutrient deficiency,particularly under conditions of nitrogen deficiency, increased yieldrelative to control plants grown under comparable conditions. Therefore,according to the present invention, there is provided a method forincreasing yield in plants grown under conditions of nutrientdeficiency, which method comprises modulating expression in a plant of anucleic acid encoding a TRY-like polypeptide. Nutrient deficiency mayresult from a lack of nutrients such as nitrogen, phosphates and otherphosphorous-containing compounds, potassium, calcium, magnesium,manganese, iron and boron, amongst others. In another embodiment of theinvention, the improved yield related traits are obtained underconditions of nitrogen deficiency.

Concerning BZR polypeptides, performance of the methods of the inventiongives plants grown under conditions of nutrient deficiency, particularlyunder conditions of nitrogen deficiency, increased seed yield relativeto control plants grown under comparable conditions. Therefore,according to the present invention, there is provided a method forincreasing seed yield in plants grown under conditions of nutrientdeficiency, which method comprises modulating expression in a plant of anucleic acid encoding a BZR polypeptide. Nutrient deficiency may resultfrom a lack of nutrients such as nitrogen, phosphates and otherphosphorous-containing compounds, potassium, calcium, cadmium,magnesium, manganese, iron and boron, amongst others.

Concerning TRY-like polypeptides, performance of the methods of theinvention gives plants grown under conditions of salt stress, increasedyield relative to control plants grown under comparable conditions.Therefore, according to the present invention, there is provided amethod for increasing yield in plants grown under conditions of saltstress, which method comprises modulating expression in a plant of anucleic acid encoding a TRY-like polypeptide. The term salt stress isnot restricted to common salt (NaCl), but may be any one or more of:NaCl, KCl, LiCl, MgCl₂, CaCl₂, amongst others.

Concerning BZR polypeptides, performance of the methods of the inventiongives plants grown under conditions of salt stress, increased seed yieldrelative to control plants grown under comparable conditions. Therefore,according to the present invention, there is provided a method forincreasing seed yield in plants grown under conditions of salt stress,which method comprises modulating expression in a plant of a nucleicacid encoding a BZR polypeptide. The term salt stress is not restrictedto common salt (NaCl), but may be any one or more of: NaCl, KCl, LiCl,MgCl₂, CaCl₂, amongst others.

The present invention encompasses plants or parts thereof (includingseeds) or cells thereof obtainable by the methods according to thepresent invention. The plants or parts thereof comprise a nucleic acidtransgene encoding a RHL1 polypeptide, or a TGase polypeptide, or aTRY-like polypeptide, or a BZR polypeptide, as defined above, operablylinked to a promoter functioning in plants.

The invention also provides genetic constructs and vectors to facilitateintroduction and/or expression in plants of nucleic acids sequencesencoding RHL1 polypeptides, or TGase polypeptides, or TRY-likepolypeptides, or BZR polypeptides. The gene constructs may be insertedinto vectors, which may be commercially available, suitable fortransforming into plants and suitable for expression of the gene ofinterest in the transformed cells. The invention also provides use of agene construct as defined herein in the methods of the invention.

More specifically, the present invention provides a constructcomprising:

-   -   (a) a nucleic acid encoding a RHL1 polypeptide, or a TGase        polypeptide, or a TRY-like polypeptide, or a BZR polypeptide, as        defined above;    -   (b) one or more control sequences capable of driving, or        increasing expression of the nucleic acid sequence of (a); and        optionally    -   (c) a transcription termination sequence.

Preferably, the nucleic acid sequence encoding a RHL1 polypeptide, or aTGase polypeptide, or a TRY-like polypeptide, or a BZR polypeptide, isas defined above. The term “control sequence” and “termination sequence”are as defined herein.

Concerning TGase polypeptides, preferably, one of the control sequencesof a construct is a seed-specific promoter isolated from a plant genome.An example of a seed-specific promoter is an alpha-globulin promoter,preferably a rice alpha-globulin promoter, more preferably analpha-globulin promoter as represented by SEQ ID NO: 72. Alternatively,a control sequence is a constitutive promoter, for example a GOS2promoter, preferably a GOS2 promoter from rice, most preferably a GOS2sequence as represented by SEQ ID NO: 71.

Plants are transformed with a vector comprising any of the nucleic acidsdescribed above. The skilled artisan is well aware of the geneticelements that must be present on the vector in order to successfullytransform, select and propagate host cells containing the sequence ofinterest. The sequence of interest is operably linked to one or morecontrol sequences (at least to a promoter).

Concerning RHL1 polypeptides, advantageously, any type of promoter,whether natural or synthetic, may be used to drive expression of thenucleic acid sequence, but preferably the promoter is of plant origin. Aconstitutive promoter is particularly useful in the methods. Preferablythe constitutive promoter is also a ubiquitous promoter. See the“Definitions” section herein for definitions of the various promotertypes. Also useful in the methods of the invention is a root-specificpromoter.

Concerning TGase polypeptides, advantageously, any type of promoter,whether natural or synthetic, may be used to increase expression of thenucleic acid sequence. A constitutive promoter is particularly useful inthe methods, preferably a constitutive promoter isolated from a plantgenome. The plant constitutive promoter drives expression of a codingsequence at a level that is in all instances below that obtained underthe control of a 35S CaMV viral promoter. An example of such a promoteris a GOS2 promoter as represented by SEQ ID NO: 71. Organ-specificpromoters, for example for preferred expression in leaves, stems,tubers, meristems, are useful in performing the methods of theinvention. Developmentally-regulated and inducible promoters are alsouseful in performing the methods of the invention. Preferably,seed-specific promoters are particularly useful in the methods of theinvention. See the “Definitions” section herein for definitions of thevarious promoter types.

Concerning TRY-like polypeptides, advantageously, any type of promoter,whether natural or synthetic, may be used to drive expression of thenucleic acid sequence, but preferably the promoter is of plant origin. Aconstitutive promoter is particularly useful in the methods. Preferablythe constitutive promoter is a ubiquitous constitutive promoter ofmedium strength. See the “Definitions” section herein for definitions ofthe various promoter types. Also useful in the methods of the inventionis a root-specific promoter.

Concerning BZR polypeptides, advantageously, any type of promoter,whether natural or synthetic, may be used to drive expression of thenucleic acid sequence, but preferably the promoter is of plant origin. Aconstitutive promoter is particularly useful in the methods.

Preferably the constitutive promoter is also a ubiquitous promoter ofmedium strength. See the “Definitions” section herein for definitions ofthe various promoter types.

Concerning RHL1 polypeptides, it should be clear that the applicabilityof the present invention is not restricted to the RHL1polypeptide-encoding nucleic acid represented by SEQ ID NO: 1, nor isthe applicability of the invention restricted to expression of a RHL1polypeptide-encoding nucleic acid when driven by a constitutivepromoter, or when driven by a root-specific promoter.

The constitutive promoter is preferably a medium strength promoter, suchas a GOS2 promoter, preferably the promoter is a GOS2 promoter fromrice. Further preferably the constitutive promoter is represented by anucleic acid sequence substantially similar to SEQ ID NO: 39, mostpreferably the constitutive promoter is as represented by SEQ ID NO: 39.See Table 2 in the “Definitions” section herein for further examples ofconstitutive promoters.

According to another preferred feature of the invention, the nucleicacid encoding an polypeptide is operably linked to a root-specificpromoter. The root-specific promoter is preferably an RCc3 promoter(Plant Mol Biol. 1995 January; 27(2):237-48), more preferably the RCc3promoter is from rice, further preferably the RCc3 promoter isrepresented by a nucleic acid sequence substantially similar to SEQ IDNO: 43, most preferably the promoter is as represented by SEQ ID NO: 43.Examples of other root-specific promoters which may also be used toperform the methods of the invention are shown in Table 3 in the“Definitions” section above.

Concerning TGase polypeptides, it should be clear that the applicabilityof the present invention is not restricted to a nucleic acid sequenceencoding the TGase polypeptide, as represented by SEQ ID NO: 45, nor isthe applicability of the invention restricted to expression of a TGasepolypeptide-encoding nucleic acid sequence when driven by aseed-specific promoter.

Concerning TRY-like polypeptides, it should be clear that theapplicability of the present invention is not restricted to the TRY-likepolypeptide-encoding nucleic acid represented by SEQ ID NO: 75, nor isthe applicability of the invention restricted to expression of aTRY-like polypeptide-encoding nucleic acid when driven by a constitutivepromoter, or when driven by a root-specific promoter.

The constitutive promoter is preferably a medium strength promoter, morepreferably selected from a plant, such as a GOS2 promoter, morepreferably is the promoter GOS2 promoter from rice. Further preferablythe constitutive promoter is represented by a nucleic acid sequencesubstantially similar to SEQ ID NO: 237, most preferably theconstitutive promoter is as represented by SEQ ID NO: 237. See the“Definitions” section herein for further examples of constitutivepromoters.

According to another preferred feature of the invention, the nucleicacid encoding a TRY-like polypeptide is operably linked to aroot-specific promoter. The root-specific promoter is preferably an RCc3promoter (Plant Mol Biol. 1995 January; 27(2):237-48), more preferablythe RCc3 promoter is from rice, further preferably the RCc3 promoter isrepresented by a nucleic acid sequence substantially similar to SEQ IDNO: 235, most preferably the promoter is as represented by SEQ ID NO:235. Examples of other root-specific promoters which may also be used toperform the methods of the invention are shown in Table 2 in the“Definitions” section above.

Concerning TRY-like polypeptides, optionally, one or more terminatorsequences may be used in the construct introduced into a plant.Preferably, the construct comprises an expression cassette essentiallysimilar or identical to SEQ ID NO 236, comprising the RCc3 promoter andthe nucleic acid encoding the TRY-like polypeptide, or an expressioncassette wherein the nucleic acid encoding the TRY-like polypeptide isoperably linked to a rice GOS2 promoter that is substantially similar toSEQ ID NO: 237.

Concerning BZR polypeptides, it should be clear that the applicabilityof the present invention is not restricted to the BZRpolypeptide-encoding nucleic acid represented by SEQ ID NO: 238, nor isthe applicability of the invention restricted to expression of a BZRpolypeptide-encoding nucleic acid when driven by a constitutivepromoter.

The constitutive promoter is preferably a medium strength promoter, morepreferably selected from a plant derived promoter, such as a GOS2promoter, more preferably is the promoter GOS2 promoter from rice.Further preferably the constitutive promoter is represented by a nucleicacid sequence substantially similar to SEQ ID NO: 322, most preferablythe constitutive promoter is as represented by SEQ ID NO: 322. See the“Definitions” section herein for further examples of plant derived andconstitutive promoters. A plant derived promoter is preferably of plantorigin. The plant derived promoter can be isolated by any of the wellknown techniques in the art from a plant or may be obtained via othersmethods such as chemical synthesis using any of the well-known suitabletechniques in the art. The plant derived promoter preferably hassubstantially the same expression pattern and strength as that of aplant promoter of plant origin. Sequence and element structure of theplant derived promoter are similar to that of a promoter of plantorigin. Preferably the plant derived promoter comprises a sequencehaving at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to a promoter of plant origin.

Optionally, one or more terminator sequences may be used in theconstruct introduced into a plant. Additional regulatory elements mayinclude transcriptional as well as translational enhancers. Thoseskilled in the art will be aware of terminator and enhancer sequencesthat may be suitable for use in performing the invention. An intronsequence may also be added to the 5′ untranslated region (UTR) or in thecoding sequence to increase the amount of the mature message thataccumulates in the cytosol, as described in the definitions section.Other control sequences (besides promoter, enhancer, silencer, intronsequences, 3′UTR and/or 5′UTR regions) may be protein and/or RNAstabilizing elements. Such sequences would be known or may readily beobtained by a person skilled in the art.

The genetic constructs of the invention may further include an origin ofreplication sequence that is required for maintenance and/or replicationin a specific cell type. One example is when a genetic construct isrequired to be maintained in a bacterial cell as an episomal geneticelement (e.g. plasmid or cosmid molecule). Preferred origins ofreplication include, but are not limited to, the f1-ori and colE1.

For the detection of the successful transfer of the nucleic acidsequences as used in the methods of the invention and/or selection oftransgenic plants comprising these nucleic acids, it is advantageous touse marker genes (or reporter genes). Therefore, the genetic constructmay optionally comprise a selectable marker gene. Selectable markers aredescribed in more detail in the “definitions” section herein. The markergenes may be removed or excised from the transgenic cell once they areno longer needed. Techniques for marker removal are known in the art,useful techniques are described above in the definitions section.

For the detection of the successful transfer of the nucleic acidsequences as used in the methods of the invention and/or selection oftransgenic plants comprising these nucleic acids, it is advantageous touse marker genes (or reporter genes). Therefore, the genetic constructmay optionally comprise a selectable marker gene. Selectable markers aredescribed in more detail in the “definitions” section herein. The markergenes may be removed or excised from the transgenic cell once they areno longer needed. Techniques for marker removal are known in the art,useful techniques are described above in the definitions section.

It is known that upon stable or transient integration of nucleic acidsequences into plant cells, only a minority of the cells takes up theforeign DNA and, if desired, integrates it into its genome, depending onthe expression vector used and the transfection technique used. Toidentify and select these integrants, a gene coding for a selectablemarker (such as the ones described above) is usually introduced into thehost cells together with the gene of interest. These markers can forexample be used in mutants in which these genes are not functional by,for example, deletion by conventional methods. Furthermore, nucleic acidsequence molecules encoding a selectable marker can be introduced into ahost cell on the same vector that comprises the sequence encoding thepolypeptides of the invention or used in the methods of the invention,or else in a separate vector. Cells which have been stably transfectedwith the introduced nucleic acid sequence can be identified for exampleby selection (for example, cells which have integrated the selectablemarker survive whereas the other cells die). The marker genes may beremoved or excised from the transgenic cell once they are no longerneeded. Techniques for marker gene removal are known in the art, usefultechniques are described above in the definitions section.

Concerning RHL1 polypeptides, or TRY-like polypeptides, the inventionalso provides a method for the production of transgenic plants havingenhanced yield-related traits relative to control plants, comprisingintroduction and expression in a plant of any nucleic acid encoding aRHL1 polypeptide, or a TRY-like polypeptide, as defined hereinabove.

Concerning TGase polypeptides, the invention also provides a method forthe production of transgenic plants having increased seed yield-relatedtraits relative to control plants, comprising introduction andexpression in a plant of any nucleic acid sequence encoding a TGasepolypeptide as defined hereinabove.

Concerning BZR polypeptides, the invention also provides a method forthe production of transgenic plants having increased seed yield relativeto control plants, comprising introduction and expression in a plant ofany nucleic acid encoding a BZR polypeptide as defined hereinabove.

Concerning RHL1 polypeptides, more specifically, the present inventionprovides a method for the production of transgenic plants havingincreased enhanced yield-related traits, particularly increased (seed)yield, which method comprises:

-   -   (i) introducing and expressing in a plant or plant cell a RHL1        polypeptide-encoding nucleic acid; and    -   (ii) cultivating the plant cell under conditions promoting plant        growth and development.

The nucleic acid of (i) may be any of the nucleic acids capable ofencoding a RHL1 polypeptide as defined herein.

Concerning TGase polypeptides, more specifically, the present inventionprovides a method for the production of transgenic plants havingincreased seed yield-related traits relative to control plants, whichmethod comprises:

-   -   (i) introducing and expressing in a plant, plant part, or plant        cell a nucleic acid sequence encoding a TGase polypeptide; and    -   (ii) cultivating the plant cell, plant part or plant under        conditions promoting plant growth and development.

The nucleic acid sequence of (i) may be any of the nucleic acidsequences capable of encoding a TGase polypeptide as defined herein.

Concerning TRY-like polypeptides, more specifically, the presentinvention provides a method for the production of transgenic plantshaving enhanced yield-related traits, particularly increased earlyvigour and/or increased seed yield, which method comprises:

-   -   (i) introducing and expressing in a plant or plant cell a        TRY-like polypeptide-encoding nucleic acid; and    -   (ii) cultivating the plant cell under conditions promoting plant        growth and development.

The nucleic acid of (i) may be any of the nucleic acids capable ofencoding a TRY-like polypeptide as defined herein.

Concerning BZR polypeptides, more specifically, the present inventionprovides a method for the production of transgenic plants havingincreased seed yield, which method comprises:

-   -   (i) introducing and expressing in a plant or plant cell a BZR        polypeptide-encoding nucleic acid; and    -   (ii) cultivating the plant cell under conditions promoting plant        growth and development.

The nucleic acid of (i) may be any of the nucleic acids capable ofencoding a BZR polypeptide as defined herein.

The nucleic acid sequence may be introduced directly into a plant cellor into the plant itself (including introduction into a tissue, organ orany other part of a plant). According to a preferred feature of thepresent invention, the nucleic acid is preferably introduced into aplant by transformation. The term “transformation” is described in moredetail in the “definitions” section herein.

The genetically modified plant cells can be regenerated via all methodswith which the skilled worker is familiar. Suitable methods can be foundin the abovementioned publications by S. D. Kung and R. Wu, Potrykus orHofgen and Willmitzer.

Generally after transformation, plant cells or cell groupings areselected for the presence of one or more markers which are encoded byplant-expressible genes co-transferred with the gene of interest,following which the transformed material is regenerated into a wholeplant. To select transformed plants, the plant material obtained in thetransformation is, as a rule, subjected to selective conditions so thattransformed plants can be distinguished from untransformed plants. Forexample, the seeds obtained in the above-described manner can be plantedand, after an initial growing period, subjected to a suitable selectionby spraying. A further possibility consists in growing the seeds, ifappropriate after sterilization, on agar plates using a suitableselection agent so that only the transformed seeds can grow into plants.Alternatively, the transformed plants are screened for the presence of aselectable marker such as the ones described above.

Following DNA transfer and regeneration, putatively transformed plantsmay also be evaluated, for instance using Southern analysis, for thepresence of the gene of interest, copy number and/or genomicorganisation. Alternatively or additionally, expression levels of thenewly introduced DNA may be monitored using Northern and/or Westernanalysis, both techniques being well known to persons having ordinaryskill in the art.

The generated transformed plants may be propagated by a variety ofmeans, such as by clonal propagation or classical breeding techniques.For example, a first generation (or T1) transformed plant may be selfedand homozygous second-generation (or T2) transformants selected, and theT2 plants may then further be propagated through classical breedingtechniques. The generated transformed organisms may take a variety offorms. For example, they may be chimeras of transformed cells andnon-transformed cells; clonal transformants (e.g., all cells transformedto contain the expression cassette); grafts of transformed anduntransformed tissues (e.g., in plants, a transformed rootstock graftedto an untransformed scion).

The present invention clearly extends to any plant cell or plantproduced by any of the methods described herein, and to all plant partsand propagules thereof. The present invention extends further toencompass the progeny of a primary transformed or transfected cell,tissue, organ or whole plant that has been produced by any of theaforementioned methods, the only requirement being that progeny exhibitthe same genotypic and/or phenotypic characteristic(s) as those producedby the parent in the methods according to the invention.

The invention also includes host cells containing an isolated nucleicacid sequence encoding a TGase polypeptide as defined hereinabove.Preferred host cells according to the invention are plant cells. Hostplants for the nucleic acid sequences or the vector used in the methodaccording to the invention, the expression cassette or construct orvector are, in principle, advantageously all plants, which are capableof synthesizing the polypeptides used in the inventive method.

The methods of the invention are advantageously applicable to any plant.Plants that are particularly useful in the methods of the inventioninclude all plants which belong to the superfamily Viridiplantae, inparticular monocotyledonous and dicotyledonous plants including fodderor forage legumes, ornamental plants, food crops, trees or shrubs.According to a preferred embodiment of the present invention, the plantis a crop plant. Examples of crop plants include soybean, sunflower,canola, alfalfa, rapeseed, linseed, cotton, tomato, potato and tobacco.Further preferably, the plant is a monocotyledonous plant. Examples ofmonocotyledonous plants include sugarcane. More preferably the plant isa cereal. Examples of cereals include rice, maize, wheat, barley,millet, rye, triticale, sorghum, emmer, spelt, secale, einkorn, teff,milo and oats.

The invention also extends to harvestable parts of a plant such as, butnot limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes,tubers and bulbs, which harvestable parts comprise a recombinant nucleicacid encoding a BZR polypeptide. The invention furthermore relates toproducts derived, preferably directly derived, from a harvestable partof such a plant, such as dry pellets or powders, oil, fat and fattyacids, starch or proteins.

According to a preferred feature of the invention, the modulatedexpression is increased expression. Methods for increasing expression ofnucleic acids or genes, or gene products, are well documented in the artand examples are provided in the definitions section.

Concerning RHL1 polypeptides, or TRY-like polypeptides, as mentionedabove, a preferred method for modulating expression of a nucleic acidsequence encoding a RHL1 polypeptide is by introducing and expressing ina plant a nucleic acid sequence encoding a RHL1 polypeptide, or aTRY-like polypeptide; however the effects of performing the method, i.e.enhancing yield-related traits may also be achieved using other wellknown techniques, including but not limited to T-DNA activation tagging,TILLING, homologous recombination. A description of these techniques isprovided in the definitions section.

Concerning TGase polypeptides, as mentioned above, a preferred methodfor increasing expression of a nucleic acid sequence encoding a TGasepolypeptide is by introducing and expressing in a plant a nucleic acidsequence encoding a TGase polypeptide; however the effects of performingthe method, i.e. increasing seed yield-related traits, may also beachieved using other well known techniques, including but not limited toT-DNA activation tagging, TILLING, homologous recombination. Adescription of these techniques is provided in the definitions section.

Concerning BZR polypeptides, as mentioned above, a preferred method formodulating expression of a nucleic acid sequence encoding a BZRpolypeptide is by introducing and expressing in a plant a nucleic acidsequence encoding a BZR polypeptide; however the effects of performingthe method, i.e. increasing seed yield may also be achieved using otherwell known techniques, including but not limited to T-DNA activationtagging, TILLING, homologous recombination. A description of thesetechniques is provided in the definitions section.

The present invention also encompasses use of nucleic acid sequencesencoding RHL1 polypeptides as described herein and use of these RHL1polypeptides in enhancing any of the aforementioned yield-related traitsin plants.

Furthermore, the present invention also encompasses use of nucleic acidsequences encoding TGase polypeptides as described herein and use ofthese TGase polypeptides in increasing any of the aforementioned seedyield-related traits in plants, under normal growth conditions, underabiotic stress growth (preferably osmotic stress growth conditions)conditions, and under growth conditions of reduced nutrientavailability, preferably under conditions of reduced nitrogenavailability.

Even furthermore, the present invention also encompasses use of nucleicacids encoding TRY-like polypeptides as described herein and use ofthese TRY-like polypeptides in enhancing any of the aforementionedyield-related traits in plants.

Furthermore, the present invention also encompasses use of nucleic acidsencoding BZR polypeptides as described herein and use of these BZRpolypeptides in increasing any of the aforementioned seed yieldparameters in plants.

Concerning RHL1 polypeptides, nucleic acid sequences encoding RHL1polypeptide described herein, or the RHL1 polypeptides themselves, mayfind use in breeding programmes in which a DNA marker is identifiedwhich may be genetically linked to a RHL1 polypeptide-encoding gene. Thenucleic acid sequences/genes, or the RHL1 polypeptides themselves may beused to define a molecular marker. This DNA or protein marker may thenbe used in breeding programmes to select plants having enhancedyield-related traits as defined hereinabove in the methods of theinvention.

Concerning TGase polypeptides, nucleic acid sequences encoding TGasepolypeptides described herein, or the TGase polypeptides themselves, mayfind use in breeding programmes in which a DNA marker is identified thatmay be genetically linked to a TGase polypeptide-encoding gene. Thegenes/nucleic acid sequences, or the TGase polypeptides themselves maybe used to define a molecular marker. This DNA or protein marker maythen be used in breeding programmes to select plants having increasedseed yield-related traits, as defined hereinabove in the methods of theinvention.

Concerning TRY-like polypeptides, nucleic acid sequences encodingTRY-like polypeptide described herein, or the TRY-like polypeptidesthemselves, may find use in breeding programmes in which a DNA marker isidentified which may be genetically linked to a TRY-likepolypeptide-encoding gene. The nucleic acid sequences/genes, or theTRY-like polypeptides themselves may be used to define a molecularmarker. This DNA or protein marker may then be used in breedingprogrammes to select plants having enhanced yield-related traits asdefined hereinabove in the methods of the invention.

Concerning BZR polypeptides, nucleic acid sequences encoding BZRpolypeptide described herein, or the BZR polypeptides themselves, mayfind use in breeding programmes in which a DNA marker is identifiedwhich may be genetically linked to a BZR polypeptide-encoding gene. Thenucleic acid sequences/genes, or the BZR polypeptides themselves may beused to define a molecular marker. This DNA or protein marker may thenbe used in breeding programmes to select plants having increased seedyield as defined hereinabove in the methods of the invention.

Allelic variants of a nucleic acid/gene encoding a RHL1 polypeptide, ora TGase polypeptide, or a TRY-like polypeptide, or a BZR polypeptide,may also find use in marker-assisted breeding programmes. Such breedingprogrammes sometimes require introduction of allelic variation bymutagenic treatment of the plants, using for example EMS mutagenesis;alternatively, the programme may start with a collection of allelicvariants of so called “natural” origin caused unintentionally.Identification of allelic variants then takes place, for example, byPCR. This is followed by a step for selection of superior allelicvariants of the sequence in question and which give increased yield.Selection is typically carried out by monitoring growth performance ofplants containing different allelic variants of the sequence inquestion. Growth performance may be monitored in a greenhouse or in thefield. Further optional steps include crossing plants in which thesuperior allelic variant was identified with another plant. This couldbe used, for example, to make a combination of interesting phenotypicfeatures.

Nucleic acid sequences encoding RHL1 polypeptides, or TGasepolypeptides, or TRY-like polypeptides, or BZR polypeptides, may also beused as probes for genetically and physically mapping the genes thatthey are a part of, and as markers for traits linked to those genes.Such information may be useful in plant breeding in order to developlines with desired phenotypes. Such use of nucleic acid sequencesencoding a RHL1 polypeptide, or a TGase polypeptide, or a TRY-likepolypeptide, or a BZR polypeptide, requires only a nucleic acid sequenceof at least 15 nucleotides in length. The nucleic acid sequencesencoding a RHL1 polypeptide, or a TGase polypeptide, or a TRY-likepolypeptide, or a BZR polypeptide, may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Sambrook J, FritschE F and Maniatis T (1989) Molecular Cloning, A Laboratory Manual) ofrestriction-digested plant genomic DNA may be probed with the nucleicacid sequences encoding a RHL1 polypeptide, or a TGase polypeptide, or aTRY-like polypeptide, or a BZR polypeptide. The resulting bandingpatterns may then be subjected to genetic analyses using computerprograms such as MapMaker (Lander et al. (1987) Genomics 1: 174-181) inorder to construct a genetic map. In addition, the nucleic acidsequences may be used to probe Southern blots containing restrictionendonuclease-treated genomic DNAs of a set of individuals representingparent and progeny of a defined genetic cross. Segregation of the DNApolymorphisms is noted and used to calculate the position of the nucleicacid sequence encoding a RHL1 polypeptide, or a TGase polypeptide, or aTRY-like polypeptide, or a BZR polypeptide, in the genetic mappreviously obtained using this population (Botstein et al. (1980) Am. J.Hum. Genet. 32: 314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4: 37-41. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

The nucleic acid sequence probes may also be used for physical mapping(i.e., placement of sequences on physical maps; see Hoheisel et al. In:Non-mammalian Genomic Analysis: A Practical Guide, Academic press 1996,pp. 319-346, and references cited therein).

In another embodiment, the nucleic acid sequence probes may be used indirect fluorescence in situ hybridisation (FISH) mapping (Trask (1991)Trends Genet. 7:149-154). Although current methods of FISH mappingfavour use of large clones (several kb to several hundred kb; see Laanet al. (1995) Genome Res. 5:13-20), improvements in sensitivity mayallow performance of FISH mapping using shorter probes.

A variety of nucleic acid sequence amplification-based methods forgenetic and physical mapping may be carried out using the nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med 11:95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleicacid sequence Res. 18:3671), Radiation Hybrid Mapping (Walter et al.(1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989)Nucleic acid sequence Res. 17:6795-6807). For these methods, thesequence of a nucleic acid sequence is used to design and produce primerpairs for use in the amplification reaction or in primer extensionreactions. The design of such primers is well known to those skilled inthe art. In methods employing PCR-based genetic mapping, it may benecessary to identify DNA sequence differences between the parents ofthe mapping cross in the region corresponding to the instant nucleicacid sequence. This, however, is generally not necessary for mappingmethods.

The methods according to the present invention result in plants havingenhanced yield-related traits, as described hereinbefore. These traitsmay also be combined with other economically advantageous traits, suchas further yield-enhancing traits, tolerance to other abiotic and bioticstresses, traits modifying various architectural features and/orbiochemical and/or physiological features.

Furthermore, the methods according to the present invention also resultin plants having increased seed yield-related traits, as describedhereinbefore. These traits may also be combined with other economicallyadvantageous traits, such as further yield-increasing traits, toleranceto abiotic and biotic stresses, tolerance to herbicides, insectides,traits modifying various architectural features and/or biochemicaland/or physiological features.

Even furthermore, the methods according to the present invention alsoresult in plants having increased seed yield, as described hereinbefore.These traits may also be combined with other economically advantageoustraits, such as further yield-enhancing traits, tolerance to otherabiotic and biotic stresses, traits modifying various architecturalfeatures and/or biochemical and/or physiological features.

Items

-   1. A method for enhancing yield-related traits in plants relative to    control plants, comprising modulating expression in a plant of a    nucleic acid encoding a Root Hairless polypeptide and optionally    selecting for plants having enhanced yield-related traits.-   2. Method according to item 1, wherein said Root Hairless    polypeptide comprises any one or more of the following motifs:

(i) Motif 9: (SEQ ID NO: 37)(SN)VMC(ED)D(YV)F(DE)(NS)(ML)(IV)VFS(DE)AWWIG(TR) K(ED)ENPEE;(ii) Motif 10: (SEQ ID NO: 38)L(AILV)A(PA)(IVA)(SA)GG(KR)(IVF)G(ED)L(KA)DL(GDS) (TS)KNP(IVL)LYLDFPQ;(iii) Motif 11: (SEQ ID NO: 39) G(RQ)(ML)KLFGTI(VL)YPKN(RK)Y(LI)TLQF;

-   -   Wherein the amino acids between brackets are alternative amino        acids at that position, and wherein in increasing order of        preference 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids are        substituted by any other amino acid, preferably by a        conservative amino acid

-   3. Method according to item 1 or 2 wherein said modulated expression    is effected by introducing and expressing in a plant a nucleic acid    encoding an Root Hairless polypeptide.

-   4. Method according to any preceding item, wherein said nucleic acid    encoding an Root Hairless polypeptide encodes any one of the    proteins listed in Table A1 or is a portion of such a nucleic acid,    or a nucleic acid capable of hybridising with such a nucleic acid or    the complement thereof.

-   5. Method according to any preceding item, wherein said nucleic acid    sequence encodes an orthologue or paralogue of any of the proteins    given in Table A1.

-   6. Method according to any preceding item, wherein said enhanced    yield-related traits comprise increased seed yield relative to    control plants.

-   7. Method according to any preceding item wherein said enhanced    yield-related traits are obtained under cultivation conditions of    nitrogen deficiency.

-   8. Method according to any one of items 3 to 7, wherein said nucleic    acid is operably linked to a constitutive promoter, preferably to a    GOS2 promoter, most preferably to a GOS2 promoter from rice.

-   9. Method according to any preceding item, wherein said nucleic acid    encoding an Root Hairless polypeptide is of plant origin, preferably    from a dicotyledonous plant, further preferably from the family    Brassicaceae, most preferably from Arabidopsis thaliana.

-   10. Plant or part thereof, including seeds, obtainable by a method    according to any preceeding item, wherein said plant or part thereof    comprises a recombinant nucleic acid encoding an Root Hairless    polypeptide.

-   11. Construct comprising:    -   (i) nucleic acid encoding a Root Hairless polypeptide as defined        in items 1, 2 or 3;    -   (ii) one or more control sequences capable of driving expression        of the nucleic acid sequence of (a); and optionally    -   (iii) a transcription termination sequence.

-   12. Construct according to item 11, wherein one of said control    sequences is a constitutive promoter, preferably a GOS2 promoter,    most preferably a GOS2 promoter from rice.

-   13. Use of a construct according to item 11 or 12 in a method for    making plants having increased yield, particularly increased seed    yield relative to control plants.

-   14. Plant, plant part or plant cell transformed with a construct    according to item 11 or 12.

-   15. Method for the production of a transgenic plant having increased    yield, preferably increased seed yield relative to control plants,    comprising:    -   (a) introducing and expressing in a plant a nucleic acid        encoding an Root Hairless polypeptide as defined in item 1 or 2;        and    -   (b) cultivating the plant cell under conditions promoting plant        growth and development; and optionally    -   (c) selecting for plants having enhanced yield-related traits

-   16. Transgenic plant having increased yield, particularly increased    biomass, relative to control plants, resulting from modulated    expression of a nucleic acid encoding an Root Hairless polypeptide    as defined in item 1 or 2 or a transgenic plant cell derived from    said transgenic plant.

-   17. Transgenic plant according to item 10, 14 or 16, or a transgenic    plant cell derived thereof, wherein said plant is a crop plant or a    monocot or a cereal, such as rice, maize, wheat, barley, millet,    rye, triticale, sorghum and oats.

-   18. Harvestable parts of a plant according to item 17, wherein said    harvestable parts are preferably shoot biomass and/or seeds.

-   19. Products derived from a plant according to item 17 and/or from    harvestable parts of a plant according to item 18.

-   20. Use of a nucleic acid encoding a Root Hairless polypeptide in    increasing yield, particularly in increasing shoot and/or biomass in    plants, relative to control plants.

-   21. A method for increasing seed yield-related traits in plants    relative to control plants, comprising increasing expression in a    plant of a nucleic acid sequence encoding a transglutaminase (TGase)    polypeptide, which TGase polypeptide comprises (i) a plastidic    transit peptide; (ii) in increasing order of preference at least    50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more    amino acid sequence identity to a domain comprising at least one    coiled coil as represented by SEQ ID NO: 27; (iii) and an Integrated    relational Enzyme database entry EC 2.3.2.13 for protein-glutamine    γ-glutamyltransferase.

-   22. Method according to item 21, wherein said TGase polypeptide    has (i) a plastidic transit peptide; (ii) in increasing order of    preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,    95%, 98%, 99% or more amino acid sequence identity to a polypeptide    as represented by SEQ ID NO: 45.

-   23. Method according to item 21 or 22, wherein said TGase    polypeptide has in increasing order of preference at least 50%, 55%,    60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid    sequence identity to a TGase polypeptide as represented by SEQ ID    NO: 45 or to any of the polypeptide sequences given in Table A2    herein.

-   24. Method according to any one of items 21 to 23, wherein said    TGase polypeptide is any polypeptide sequence which when used in the    construction of a TGase phylogenetic tree, such as the one depicted    in FIG. 5, clusters with the clade of TGase polypeptides comprising    the polypeptide sequence as represented by SEQ ID NO: 45, rather    than with the other clades.

-   25. Method according to any one of items 21 to 24, wherein said    TGase polypeptide is a polypeptide with enzymatic activity    consisting in catalyzing the formation of amide linkages, generally    in a Ca-dependent fashion, between the primary amine of an amine    donor substrate and the y-carboxamide group of peptide-bound    endo-glutamine residues in proteins or polypeptides that are the    amine acceptors.

-   26. Method according to any one of items 21 to 25, wherein said    nucleic acid sequence encoding a TGase polypeptide is represented by    any one of the nucleic acid sequence SEQ ID NOs given in Table A or    a portion thereof, or a sequence capable of hybridising with any one    of the nucleic acid sequences SEQ ID NOs given in Table A2, or to a    complement thereof.

-   27. Method according to any one of items 21 to 26, wherein said    nucleic acid sequence encodes an orthologue or paralogue of any of    the polypeptide sequence SEQ ID NOs given in Table A2.

-   28. Method according to any one of items 21 to 27, wherein said    increased expression is effected by any one or more of: T-DNA    activation tagging, TILLING, or homologous recombination.

-   29. Method according to any one of items 21 to 28, wherein said    increased expression is effected by introducing and expressing in a    plant a nucleic acid sequence encoding a TGase polypeptide.

-   30. Method according to any one of items 21 to 29, wherein said    increased seed yield-related trait is one or more of: increased    total seed yield per plant, increased number of filled seeds, and    increased harvest index.

-   31. Method according to any one of items 21 to 30, wherein said    nucleic acid sequence is operably linked to a seed-specific    promoter.

-   32. Method according to item 31, wherein said seed-specific promoter    is an alpha-globulin promoter, preferably a rice alpha-globulin    promoter, more preferably an alpha-globulin promoter as represented    by SEQ ID NO: 72.

-   33. Method according to any one of items 21 to 32, wherein said    nucleic acid sequence encoding a TGase polypeptide is from a plant,    further preferably from a monocotyledonous plant, more preferably    from the family Poaceae, most preferably the nucleic acid sequence    is from Oryza sativa.

-   34. Plants, parts thereof (including seeds), or plant cells    obtainable by a method according to any one of items 21 to 33,    wherein said plant, part or cell thereof comprises an isolated    nucleic acid transgene encoding a TGase polypeptide.

-   35. Construct comprising:    -   (a) a nucleic acid sequence encoding a TGase polypeptide as        defined in any one of items 21 to 27;    -   (b) one or more control sequences capable of driving expression        of the nucleic acid sequence of (a); and optionally    -   (c) a transcription termination sequence.

-   36. Construct according to item 35, wherein said control sequence is    a seed-specific promoter.

-   37. Construct according to item 36, wherein said seed-specific    promoter is an alpha-globulin promoter, preferably a rice    alpha-globulin promoter, more preferably an alpha-globulin promoter    as represented by SEQ ID NO: 72.

-   38. Use of a construct according to any one of items 35 to 37, in a    method for making plants having increased seed yield-related traits    relative to control plants, which increased seed yield-related    traits are one or more of: increased total seed yield per plant,    increased number of filled seeds, and increased harvest index.

-   39. Plant, plant part or plant cell transformed with a construct    according to any one of items 35 to 37.

-   40. Method for the production of transgenic plants having increased    seed yield-related traits relative to control plants, comprising:    -   (i) introducing and expressing in a plant, plant part, or plant        cell, a nucleic acid sequence encoding a TGase polypeptide as        defined in any one of items 1 to 7; and    -   (ii) cultivating the plant cell, plant part, or plant under        conditions promoting plant growth and development.

-   41. Transgenic plant having increased seed yield-related traits    relative to control plants, resulting from increased expression of    an isolated nucleic acid sequence encoding a TGase polypeptide as    defined in any one of items 21 to 27, or a transgenic plant cell or    transgenic plant part derived from said transgenic plant.

-   42. Transgenic plant according to item 34, 39 or 41, wherein said    plant is a crop plant or a monocot or a cereal, such as rice, maize,    wheat, barley, millet, rye, triticale, sorghum and oats, or a    transgenic plant cell derived from said transgenic plant.

-   43. Harvestable parts comprising an isolated nucleic acid sequence    encoding a TGase polypeptide, of a plant according to item 42,    wherein said harvestable parts are preferably seeds.

-   44. Products derived from a plant according to item 42 and/or from    harvestable parts of a plant according to item 43.

-   45. Use of a nucleic acid sequence encoding a TGase polypeptide as    defined in any one of items 21 to 27 in increasing seed    yield-related traits, comprising one or more of: increased total    seed yield per plant, increased number of filled seeds, and    increased harvest index.

-   46. A method for enhancing yield-related traits in plants relative    to control plants, comprising modulating expression in a plant of a    nucleic acid encoding a TRY-like polypeptide, wherein said TRY-like    polypeptide comprises a Myb-like DNA-binding domain (Panther    PTHR10641:SF26; Gene3D G3DSA:1.10.10.60).

-   47. Method according to item 46, wherein said TRY-like polypeptide    comprises one or more of motifs 12 to 15 (SEQ ID NO: 229 to SEQ ID    NO: 232).

-   48. Method according to item 46 or 47, wherein said modulated    expression is effected by introducing and expressing in a plant a    nucleic acid encoding a TRY-like polypeptide.

-   49. Method according to any one of items 46 to 48, wherein said    nucleic acid encoding a TRY-like polypeptide encodes any one of the    proteins listed in Table A3 or is a portion of such a nucleic acid,    or a nucleic acid capable of hybridising with such a nucleic acid.

-   50. Method according to any one of items 46 to 49, wherein said    nucleic acid sequence encodes an orthologue or paralogue of any of    the proteins given in Table A3.

-   51. Method according to any one of items 46 to 50, wherein said    enhanced yield-related traits comprise increased emergence vigour    and/or increased yield, relative to control plants.

-   52. Method according to any one of items 46 to 51, wherein said    enhanced yield-related traits are obtained under non-stress    conditions or under conditions of nitrogen deficiency.

-   53. Method according to any one of items 48 to 52, wherein said    nucleic acid is operably linked to a root-specific promoter,    preferably to a RCc3 promoter, most preferably to a RCc3 promoter    from rice, or wherein said nucleic acid is operably linked to a    constitutive promoter, preferably to a GOS2 promoter, most    preferably to a GOS2 promoter from rice.

-   54. Method according to any one of items 46 to 53, wherein said    nucleic acid encoding a TRY-like polypeptide is of plant origin,    preferably from a dicotyledonous plant, further preferably from the    family Brassicaceae, more preferably from the genus Arabidopsis,    most preferably from Arabidopsis thaliana.

-   55. Plant or part thereof, including seeds, obtainable by a method    according to any one of items 46 to 48, wherein said plant or part    thereof comprises a recombinant nucleic acid encoding a TRY-like    polypeptide.

-   56. Construct comprising:    -   (a) nucleic acid encoding a TRY-like polypeptide as defined in        items 1 or 2;    -   (b) one or more control sequences capable of driving expression        of the nucleic acid sequence of (a); and optionally    -   (c) a transcription termination sequence.

-   57. Construct according to item 56, wherein one of said control    sequences is a constitutive promoter, preferably a RCc3 promoter,    most preferably a RCc3 promoter from rice, or wherein one of said    control sequences is a constitutive promoter, preferably a RCc3    promoter, most preferably a RCc3 promoter from rice.

-   58. Use of a construct according to item 56 or 57 in a method for    making plants having increased yield-related traits, particularly    increased emergence vigour and/or increased seed yield relative to    control plants.

-   59. Plant, plant part or plant cell transformed with a construct    according to item 56 or 57.

-   60. Method for the production of a transgenic plant having increased    yield-related traits, particularly increased emergence vigour and/or    increased seed yield relative to control plants, comprising:    -   (i) introducing and expressing in a plant a nucleic acid        encoding a TRY-like polypeptide as defined in item 1 or 2; and    -   (ii) cultivating the plant cell under conditions promoting plant        growth and development.

-   61. Transgenic plant having increased emergence vigour and/or    increased yield, relative to control plants, resulting from    modulated expression of a nucleic acid encoding a TRY-like    polypeptide as defined in item 46 or 47, or a transgenic plant cell    derived from said transgenic plant.

-   62. Transgenic plant according to item 55, 59 or 61, or a transgenic    plant cell derived thereof, wherein said plant is a crop plant or a    monocot or a cereal, such as rice, maize, wheat, barley, millet,    rye, triticale, sorghum emmer, spelt, secale, einkorn, teff, milo    and oats.

-   63. Harvestable parts of a plant according to item 62, wherein said    harvestable parts are preferably seeds.

-   64. Products derived from a plant according to item 62 and/or from    harvestable parts of a plant according to item 63.

-   65. Use of a nucleic acid encoding a TRY-like polypeptide in    increasing emergence vigour and/or increasing yield in plants,    relative to control plants.

-   66. A method for increasing seed yield in plants relative to control    plants, comprising modulating expression in a plant of a nucleic    acid encoding a BZR, BRASSINAZOLE-RESISTANT polypeptide and    optionally selecting for plants having increased seed yield.

-   67. Method according to item 66, wherein said BZR polypeptide    comprises:    -   (i) a domain having in increasing order of preference at least        50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,        63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,        76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,        89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%        sequence identity to the domain located between amino acid        coordinates 10-157 in SEQ ID NO: 239 and/or    -   (ii) a domain having in increasing order of preference at least        50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,        63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,        76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,        89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%        sequence identity to a bHLH-like domain as represented SEQ ID        NO: 326 and/or    -   (iii) 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%,        85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,        98%, 99% or more sequence identity to the amino acid sequence of        any of the polypeptides of Table A4; and/or    -   (iv) a motif as represented by any one of SEQ ID NO: 323, SEQ ID        NO: 324, SEQ ID NO: 325, wherein 1, 2, 3 or 4 residues may be        substituted by any amino acid.

-   68. Method according to item 66 or 67, wherein said modulated    expression is effected by introducing and expressing in a plant a    nucleic acid encoding a BZR polypeptide.

-   69. Method according to any one of items 66 to 68, wherein said    nucleic acid encoding a BZR polypeptide encodes any one of the    proteins listed in Table A4 or is a portion of such a nucleic acid,    or a nucleic acid capable of hybridising with such a nucleic acid.

-   70. Method according to any preceding item, wherein said nucleic    acid sequence encodes an orthologue or paralogue of any of the    proteins given in Table A4.

-   71. Method according to any one of items 66 to 70, wherein said    increased seed yield is selected from the total weight of the seed,    the number of filled seed and the thousand kernel weight.

-   72. Method according to any one of items 66 to 71, wherein said    increased seed yield is obtained under non-stress conditions.

-   73. Method according to any one of items 68 to 72, wherein said    nucleic acid is operably linked to a plant derived constitutive    promoter, preferably to a GOS2 promoter, most preferably to a GOS2    promoter from rice.

-   74. Method according to any one of items 66 to 73, wherein said    nucleic acid encoding a

BZR polypeptide is of plant origin, preferably from a dicotyledonousplant, further preferably from the family Brasicaceae, most preferablyfrom Arabidopsis thaliana.

-   75. Plant or part thereof, including seeds, obtainable by a method    according to any one of items 66 to 74, wherein said plant or part    thereof comprises a recombinant nucleic acid encoding a BZR    polypeptide.-   76. An isolated nucleic acid molecule comprising any one of the    following features:    -   (i) a nucleic acid represented by any one of SEQ ID NO: 250,        252, 254, 256;    -   (ii) a nucleic acid or fragment thereof that is complementary to        any one of SEQ ID NO: 250, 252, 254, 256;    -   (iii) a nucleic acid encoding a BZR polypeptide having, in        increasing order of preference, at least 80%, 81%, 82%, 83%,        84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,        97%, 98%, or 99% or more sequence identity to one of SEQ ID NO:        252, 254, 256, 258;    -   (iv) a nucleic acid capable of hybridizing under stringent        conditions to any one of the nucleic acids given in (i), (ii)        or (iii) above.-   77. An isolated polypeptide comprising:    -   (i) an amino acid sequence having, in increasing order of        preference, at least at least 80%, 81%, 82%, 83%, 84%, 85%, 86%,        87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or        99% or more sequence identity to one of SEQ ID NO: 252, 254,        256, 258; and/or    -   (ii) derivatives of any of the amino acid sequences given in        (i).-   78. Construct comprising:    -   (i) nucleic acid encoding a BZR polypeptide as defined in items        66, 67 or 77, or a nucleic acid according to item 76;    -   (ii) one or more control sequences capable of driving expression        of the nucleic acid sequence of (a); and optionally    -   (iii) a transcription termination sequence.-   79. Construct according to item 78, wherein one of said control    sequences is a constitutive promoter, preferably a GOS2 promoter,    most preferably a GOS2 promoter from rice.-   80. Use of a construct according to item 13 or 14 in a method for    making plants having increased seed yield relative to control    plants.-   81. Plant, plant part or plant cell transformed with a construct    according to item 78 or 79.-   82. Method for the production of a transgenic plant having increased    seed yield relative to control plants, comprising:    -   (i) introducing and expressing in a plant a nucleic acid        encoding a BZR polypeptide as defined in item 66, 67 or 77, or a        nucleic acid according to item 11; and    -   (ii) cultivating the plant cell under conditions promoting plant        growth and development;

and optionally

-   -   (iii) selecting for plants having seed yield.

-   83. Transgenic plant having increased seed yield resulting from    modulated expression of a nucleic acid encoding a BZR polypeptide as    defined in item 66, 67 or 77 or a transgenic plant cell derived from    said transgenic plant.

-   84. Transgenic plant according to item 75, 81 or 83, or a transgenic    plant cell derived thereof, wherein said plant is a crop plant or a    monocot or a cereal, such as rice, maize, wheat, barley, millet,    rye, triticale, sorghum and oats.

-   85. Harvestable parts of a plant according to item 84, wherein said    harvestable parts are preferably shoot biomass and/or seeds.

-   86. Products derived from a plant according to item 85 and/or from    harvestable parts of a plant according to item 85.

-   87. Use of a nucleic acid encoding a BZR polypeptide in increasing    seed yield relative to control plants.

DESCRIPTION OF FIGURES

The present invention will now be described with reference to thefollowing figures in which:

FIG. 1 represents the sequence of SEQ ID NO: 2 with conserved putativenuclear localization signals.

FIG. 2 represents multiple alignment of RHL1 polypeptide. O.sativa_Os07g07580: SEQ ID NO: 10; O. sativa_Os06g51380: SEQ ID NO: 12;Z. mays_TA180670: SEQ ID NO: 14; A. formosa_TA10038: SEQ ID NO: 16; V.vinifera_GSVIVT00027050001: SEQ ID NO: 28; M. domestica_TA43921: SEQ IDNO: 18; S. tuberosurn_TA36268: SEQ ID NO: 24; p.trichocarpa_scaff_IV.277: SEQ ID NO: 8; A. thaliana_AT1G48380.1: SEQ IDNO: 2; P. patens _(—)74926: SEQ ID NO: 20; and P. patens _(—)173149: SEQID NO: 22.

FIG. 3 shows phylogenetic tree of RHL1 polypeptide

FIG. 4 represents the binary vector for increased expression in Oryzasativa of a RHL1-encoding nucleic acid under the control of a rice GOS2promoter (pGOS2)

FIG. 5 shows a phylogenetic tree of TGase polypeptides from varioussource organisms, according to Villalobos et al. (2004; Gene 336:93-104). TGases useful in performing the methods of the invention(essentially from plants) are shown with a bracket, the clade split witha circle, the arrow points to the Oryza sativa TGase polypeptide asrepresented by SEQ ID NO: 45.

FIG. 6 shows the graphical output of the COILS algorithm predicting atleast one coiled coil domain in the polypeptide as represented by SEQ IDNO: 45. The X axis represents the amino acid residue coordinates, the Yaxis the probability (ranging from 0 to 1) that a coiled coil domain ispresent, and the three lines, the three windows (14, 21, 28) examined.

FIG. 7 shows an AlignX (from Vector NTI 10.3, Invitrogen Corporation)multiple sequence alignment of the TGase polypeptides from Table A2. TheN-terminal plastidic transit peptide is separated from the rest of thepolypeptide (mature polypeptide) by a vertical bar. The putative calciumbinding region is boxed, and marked with X's under the consensussequence. The domain where at least one coiled coil is predicted usingthe Coils algorithm (and as represented by SEQ ID NO: 70) is marked withX's under the consensus region. Orysa_TGase: SEQ ID NO: 45; Sorbi_TGase:SEQ ID NO: 61; Sacof_TGase: SEQ ID NO: 59; Zeama_TGase 2: SEQ ID NO: 63;Zeama_TGase 3: SEQ ID NO: 65; Zeama_tgz15: SEQ ID NO: 67; Zeama_tgz21:SEQ ID NO: 69; Poptr_TGase: SEQ ID NO: 57; Lyces_TGase: SEQ ID NO: 51;Horvu_TGase: SEQ ID NO: 49; Orysa_TGase 2: SEQ ID NO: 53; Arath_TGase:SEQ ID NO: 47; Picsi_TGAse: SEQ ID NO: 55; and Consensus: SEQ ID NO:329.

FIG. 8 shows the binary vector for increased expression in Oryza sativaplants of a nucleic acid sequence encoding a TGase polypeptide under thecontrol of a promoter functioning in plants.

FIG. 9 represents the sequence of SEQ ID NO: 76 with conserved motifs ordomains: the Myb-like DNA-binding domain as identified with HMMPfam(PF00249.17) is indicated in bold, the sequence that is covered by motif12 is underlined.

FIG. 10 represents a multiple alignment of various TRY-like polypeptidesequences. A dot indicates conserved residues, a colon indicates highlyconserved residues and an asterisk stands for perfectly conservedresidues. The highest degree of sequence conservation is found in theregion of the DNA-binding domain. A. sativa_CN818591: SEQ ID NO: 84; L.multiflorum_AU249134: SEQ ID NO: 148; A. capillaris_DV853805: SEQ ID NO:78; A. capillaris_DV859458: SEQ ID NO: 80; H. vulgare_TC189825: SEQ IDNO: 138; T. aestivum_BE412359: SEQ ID NO: 216; O. minuta_CB884361: SEQID NO: 164; O. sativa_LOC_Os01g43230.2: SEQ ID NO: 166; S.bicolor_Sb03g028170.1: SEQ ID NO: 208; Z. mays_TC409725: SEQ ID NO: 228;T. androssowii_TA2313_(—)189785: SEQ ID NO: 218; Triphysaria_sp_TC9313:SEQ ID NO: 220; S. tuberosum_CV505951: SEQ ID NO: 214; P.tremula_TA11725_(—)113636: SEQ ID NO: 192; P. trichocarpa _(—)594467:SEQ ID NO: 200; P. trichocarpa _(—)562293: SEQ ID NO: 196; B.gymnorrhiza_TA2541_(—)39984: SEQ ID NO: 102; M.esculenta_TA9427_(—)3983: SEQ ID NO: 160; P. persica_BU039343: SEQ IDNO: 176; V. vinifera_GSVIVT00026045001: SEQ ID NO: 226; L.tulipifera_CV004984: SEQ ID NO: 154; G. hirsutum_TC121748: SEQ ID NO:128; E. esula_DV121180: SEQ ID NO: 120; M. domestica_TC17597: SEQ ID NO:156; P. tremula_BU888423: SEQ ID NO: 188; P. trichocarpa _(—)568212: SEQID NO: 198; G. hirsutum_DW508052: SEQ ID NO: 122; G. hirsutum_TC116960:SEQ ID NO: 126; V. vinifera GSVIVT00006915001: SEQ ID NO: 222; J.hindsii_×_(—) regia_TA1295_(—)43229: SEQ ID NO: 144; G. max _(—)8223:SEQ ID NO: 132; P. vulgaris_CV538421: SEQ ID NO: 206; C.tetragonoloba_EG990179: SEQ ID NO: 118; G. max _(—)29139: SEQ ID NO:134; G. soja_TA4526_(—)3848: SEQ ID NO: 136; L. japonicus_CB827663: SEQID NO: 146; C. canephora_DV693718: SEQ ID NO: 114; P. hybrida_EB175070:SEQ ID NO: 172; I. nil_TC6509: SEQ ID NO: 140; L. saligna_DW052030: SEQID NO: 150; A. hypogaea_CD038483: SEQ ID NO: 82; P.tremula_TA7610_(—)113636: SEQ ID NO: 194; P. equestris_CB034844: SEQ IDNO: 168; V. vinifera_GSVIVT00010755001: SEQ ID NO: 224; P.pinaster_CT579117: SEQ ID NO: 178; P. taeda_DR096185: SEQ ID NO: 186; P.sitchensis_TA16538_(—)3332: SEQ ID NO: 182; A.thaliana_At1g01380_CPC-like_: SEQ ID NO: 86; B. napus_EV055366: SEQ IDNO: 108; A. thaliana_At4g01060_CPC-like_: SEQ ID NO: 100; A.thaliana_At2g46410_CPC-like_: SEQ ID NO: 98; B. napus_TC92601: SEQ IDNO: 110; A. thaliana_At5g53200_CPC-like_: SEQ ID NO: 76; B.napus_EE451172: SEQ ID NO: 106; J. hindsii_×_(—) regia_EL893054: SEQ IDNO: 142; G. hirsutum_TC102183: SEQ ID NO: 124; P. trichocarpa_(—)807368: SEQ ID NO: 204; M. esculenta_DV443286: SEQ ID NO: 158; P.tremula_DN497189: SEQ ID NO: 190; P. trichocarpa _(—)674550: SEQ ID NO:202; C. longa_DY390653: SEQ ID NO: 116; A. thaliana_At2g30420_CPC-like_:SEQ ID NO: 96; B. napus_CD843377: SEQ ID NO: 104; B. napus_TC95812: SEQID NO: 112; G. max_Glyma11g02060.1: SEQ ID NO: 130; M.truncatula_CT033771_(—)17.4: SEQ ID NO: 162; L. serriola_DW108811: SEQID NO: 152; S. miltiorrhiza_CV166339: SEQ ID NO: 210; S.miltiorrhiza_TA1626_(—)226208: SEQ ID NO: 212; P. glauca_DR564374: SEQID NO: 170; P. sitchensis_TA17447_(—)3332: SEQ ID NO: 184; P.pinaster_TA6535_(—)71647: SEQ ID NO: 180; P. menziesii_TA3655_(—)3357:SEQ ID NO: 174; A. thaliana_At1g71030_CPC-like_: SEQ ID NO: 94; A.thaliana_At1g18960_CPC-like_: SEQ ID NO: 90; A.thaliana_At1g09710_CPC-like_: SEQ ID NO: 88; and A.thaliana_At1g58220_CPC-like_: SEQ ID NO: 92.

FIG. 11 represents the binary vector for increased expression in Oryzasativa of a TRY-like-encoding nucleic acid under the control of a riceRCc3 promoter (pRCc3).

FIG. 12 represents a multiple alignment of BZR polypeptides.AT1G19350.1: SEQ ID NO: 241; AT1G75080.1: SEQ ID NO: 243;Pt_scaff_(—)40.175: SEQ ID NO: 295; Pt_scaff_II.1237: SEQ ID NO: 299; Gm1762729: SEQ ID NO: 251; Mt_TA28179_(—)3880: SEQ ID NO: 273; Le LAT61:SEQ ID NO: 261; Le_TA51962_(—)4081: SEQ ID NO: 267; Vv_TA44770_(—)29760:SEQ ID NO: 311; AT3G50750.1: SEQ ID NO: 239; Gm 1768381: SEQ ID NO: 255;Gm 1768507: SEQ ID NO: 257; Mt_TA21345_(—)3880: SEQ ID NO: 271;Pt_scaff_(—)57.215: SEQ ID NO: 297; Pt_scaff_VII.1038: SEQ ID NO: 303;Le_DB718708: SEQ ID NO: 263; Le_TA37112_(—)4081: SEQ ID NO: 265;Os07g0580500: SEQ ID NO: 281; Zm_AY107201: SEQ ID NO: 313; AT4G36780.1:SEQ ID NO: 249; Os02g0129600: SEQ ID NO: 277; Zm_EE158804: SEQ ID NO:315; Zm_TA189809_(—)4577: SEQ ID NO: 330; Pp 82495: SEQ ID NO: 287; Pp17189: SEQ ID NO: 283; Pp 172161: SEQ ID NO: 285; Ps WS0287_(—)023: SEQID NO: 289; Hv_TA37786_(—)4513: SEQ ID NO: 259; Os01g0203000: SEQ ID NO:275; Zm_TA178991_(—)4577: SEQ ID NO: 319; Os06g0552300: SEQ ID NO: 279;Zm_TA1750444577: SEQ ID NO: 317; AT1G78700.1: SEQ ID NO: 245;AT4G18890.1: SEQ ID NO: 247; Pt_scaff_IV.340: SEQ ID NO: 301;Pt_scaff_XI.678: SEQ ID NO: 305; Gm 1765606: SEQ ID NO: 253;Mt_BF635822: SEQ ID NO: 269; Pt WS01123_K11: SEQ ID NO: 291;Pt_scaff_(—)178.36: SEQ ID NO: 293; Pt_scaff_XI.792: SEQ ID NO: 307; SIFC26BA11: SEQ ID NO: 309; and Consensus: SEQ ID NO: 331.

FIG. 13 represents the binary vector for increased expression in Oryzasativa of a BZR-encoding nucleic acid under the control of a rice GOS2promoter (pGOS2).

EXAMPLES

The present invention will now be described with reference to thefollowing examples, which are by way of illustration alone. Thefollowing examples are not intended to completely define or otherwiselimit the scope of the invention.

DNA manipulation: unless otherwise stated, recombinant DNA techniquesare performed according to standard protocols described in (Sambrook(2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold SpringHarbor Laboratory Press, CSH, New York) or in Volumes 1 and 2 of Ausubelet al. (1994), Current Protocols in Molecular Biology, CurrentProtocols. Standard materials and methods for plant molecular work aredescribed in Plant Molecular Biology Labfax (1993) by R. D. D. Croy,published by BIOS Scientific Publications Ltd (UK) and BlackwellScientific Publications (UK).

Example 1 Identification of Sequences Related to the Nucleic AcidSequence Used in the Methods of the Invention 1.1. Root Hairless 1(RHL1)

Sequences (full length cDNA, ESTs or genomic) related to the nucleicacid sequence used in the methods of the present invention wereidentified amongst those maintained in the Entrez Nucleotides databaseat the National Center for Biotechnology Information (NCBI) usingdatabase sequence search tools, such as the Basic Local Alignment Tool(BLAST) (Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschulet al. (1997) Nucleic Acids Res. 25:3389-3402). The program is used tofind regions of local similarity between sequences by comparing nucleicacid or polypeptide sequences to sequence databases and by calculatingthe statistical significance of matches. For example, the polypeptideencoded by the nucleic acid used in the present invention was used forthe TBLASTN algorithm, with default settings and the filter to ignorelow complexity sequences set off. The output of the analysis was viewedby pairwise comparison, and ranked according to the probability score(E-value), where the score reflect the probability that a particularalignment occurs by chance (the lower the E-value, the more significantthe hit). In addition to E-values, comparisons were also scored bypercentage identity. Percentage identity refers to the number ofidentical nucleotides (or amino acids) between the two compared nucleicacid (or polypeptide) sequences over a particular length. In someinstances, the default parameters may be adjusted to modify thestringency of the search. For example the E-value may be increased toshow less stringent matches. This way, short nearly exact matches may beidentified.

Table A1 provides a list of nucleic acid sequences related to thenucleic acid sequence used in the methods of the present invention.

TABLE A1 Examples of RHL1 nucleic acids and polypeptides: Nucleic acidProtein Name Source Organism SEQ ID NO: SEQ ID NO:A.thaliana_AT1G48380.1 (Arath_RHL1) Arabidopsis thaliana 1 2p.trichocarpa_scaff_44.278 Populus trichocarpa 3 4p.trichocarpa_scaff_184.3 Populus trichocarpa 5 6p.trichocarpa_scaff_IV.277 Populus trichocarpa 7 8 O.sativa_Os07g07580(Orysa_RHL1) Oryza sativa 9 10 O.sativa_Os06g51380 Oryza sativa 11 12Z.mays_TA180670 Zea mays 13 14 A.formosa_TA10038 Aquilegia formosa 15 16M.domestica_TA43921 Malus domestica 17 18 P.patens_74926 Physcomitrellapatens 19 20 P.patens_173149 Physcomitrella patens 21 22S.tuberosum_TA36268 Solanum tuberosum 23 24 V.shuttleworthii_TA2694Vitis shuttleworthii 25 26 V.vinifera_GSVIVT00027050001 Vitis vinifera27 28

In some instances, related sequences have tentatively been assembled andpublicly disclosed by research institutions, such as The Institute forGenomic Research (TIGR). The Eukaryotic Gene Orthologs (EGO) databasemay be used to identify such related sequences, either by keyword searchor by using the BLAST algorithm with the nucleic acid or polypeptidesequence of interest.

1.2. Transglutaminases (TGases)

Sequences (full length cDNA, ESTs or genomic) related to the nucleicacid sequence used in the methods of the present invention wereidentified amongst those maintained in the Entrez Nucleotides databaseat the National Center for Biotechnology Information (NCBI) usingdatabase sequence search tools, such as the Basic Local Alignment Tool(BLAST) (Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschulet al. (1997) Nucleic Acids Res. 25:3389-3402). The program is used tofind regions of local similarity between sequences by comparing nucleicacid sequence or polypeptide sequences to sequence databases and bycalculating the statistical significance of matches. For example, thepolypeptide encoded by the nucleic acid sequence of the presentinvention was used for the TBLASTN algorithm, with default settings andthe filter to ignore low complexity sequences set off. The output of theanalysis was viewed by pairwise comparison, and ranked according to theprobability score (E-value), where the score reflect the probabilitythat a particular alignment occurs by chance (the lower the E-value, themore significant the hit). In addition to E-values, comparisons werealso scored by percentage identity. Percentage identity refers to thenumber of identical nucleotides (or amino acids) between the twocompared nucleic acid sequence (or polypeptide) sequences over aparticular length. In some instances, the default parameters may beadjusted to modify the stringency of the search. For example the E-valuemay be increased to show less stringent matches. This way, short nearlyexact matches may be identified.

Table A2 provides a list of nucleic acid sequences related to thenucleic acid sequence used in the methods of the present invention.

TABLE A2 Examples of TGase polypeptide sequences, and encoding nucleicacid sequences: Nucleic acid Polypeptide Public database sequencesequence Name Source organism accession number SEQ ID NO: SEQ ID NO:Orysa_TGase Oryza sativa na 44 45 Arath_TGase Arabidopsis thalianaNM_105387 46 47 Horvu_TGase Hordeum vulgare AK251411 48 49 Lyces_TGaseLycopersicon esculentum BT012898 50 51 Orysa_TGase II Oryza sativaNM_001052696 52 53 Picsi_TGAse Picea sitchensis EF087701 54 55Poptr_TGase Populus tremuloides TA16744_3694 56 57 Sacof_TGase Saccharumofficinarum CA246119 58 59 CA254082 CA265940 Sorbi_TGase Sorghum bicolorCL187991 60 61 ER757182.1 CW291038 Zeama_TGase II Zea mays DT641696.1,62 63 DV540831.1 Zeama_TGase III Zea mays na 64 65 Zeama_tgz15 Zea maysAJ421525 66 67 Zeama_tgz21 Zea mays AJ488103 68 69

In some instances, related sequences have tentatively been assembled andpublicly disclosed by research institutions, such as The Institute forGenomic Research (TIGR; beginning with TA). The Eukaryotic GeneOrthologs (EGO) database may be used to identify such related sequences,either by keyword search or by using the BLAST algorithm with thenucleic acid sequence or polypeptide sequence of interest. On otherinstances, special nucleic acid sequence databases have been created forparticular organisms, such as by the Joint Genome Institute.

1.3. Tryptichon (TRY-Like)

Sequences (full length cDNA, ESTs or genomic) related to the nucleicacid sequence used in the methods of the present invention wereidentified amongst those maintained in the Entrez Nucleotides databaseat the National Center for Biotechnology Information (NCBI) usingdatabase sequence search tools, such as the Basic Local Alignment Tool(BLAST) (Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschulet al. (1997) Nucleic Acids Res. 25:3389-3402). The program is used tofind regions of local similarity between sequences by comparing nucleicacid or polypeptide sequences to sequence databases and by calculatingthe statistical significance of matches. For example, the polypeptideencoded by the nucleic acid used in the present invention was used forthe TBLASTN algorithm, with default settings and the filter to ignorelow complexity sequences set off. The output of the analysis was viewedby pairwise comparison, and ranked according to the probability score(E-value), where the score reflect the probability that a particularalignment occurs by chance (the lower the E-value, the more significantthe hit). In addition to E-values, comparisons were also scored bypercentage identity. Percentage identity refers to the number ofidentical nucleotides (or amino acids) between the two compared nucleicacid (or polypeptide) sequences over a particular length. In someinstances, the default parameters may be adjusted to modify thestringency of the search. For example the E-value may be increased toshow less stringent matches. This way, short nearly exact matches may beidentified.

Table A3 provides a list of nucleic acid sequences related to thenucleic acid sequence used in the methods of the present invention.

TABLE A3 Examples of TRY-like polypeptides: Nucleic Poly- acid peptideSEQ SEQ Name ID NO: ID NO: At5g53200 75 76 A.capillaris_DV853805 77 78A.capillaris_DV859458 79 80 A.hypogaea_CD038483 81 82 A.sativa_CN81859183 84 A.thaliana_At1g01380_CPC-like_ETC1 85 86A.thaliana_At1g09710_CPC-like_NA 87 88 A.thaliana_At1g18960_CPC-like_NA89 90 A.thaliana_At1g58220_CPC-like_NA 91 92A.thaliana_At1g71030_CPC-like_ATMYBL2 93 94A.thaliana_At2g30420_CPC-like_NA 95 96 A.thaliana_At2g46410_CPC-like_CPC97 98 A.thaliana_At4g01060_CPC-like_ETC3 99 100B.gymnorrhiza_TA2541_39984 101 102 B.napus_CD843377 103 104B.napus_EE451172 105 106 B.napus_EV055366 107 108 B.napus_TC92601 109110 B.napus_TC95812 111 112 C.canephora_DV693718 113 114C.longa_DY390653 115 116 C.tetragonoloba_EG990179 117 118E.esula_DV121180 119 120 G.hirsutum_DW508052 121 122 G.hirsutum_TC102183123 124 G.hirsutum_TC116960 125 126 G.hirsutum_TC121748 127 128G.max_Glyma11g02060.1 129 130 G.max_8223 131 132 G.max_29139 133 134G.soja_TA4526_3848 135 136 H.vulgare_TC189825 137 138 I.nil_TC6509 139140 J.hindsii_x_regia_EL893054 141 142 J.hindsii_x_regia_TA1295_432290143 144 L.japonicus_CB827663 145 146 L.multiflorum_AU249134 147 148L.saligna_DW052030 149 150 L.serriola_DW108811 151 152L.tulipifera_CV004984 153 154 M.domestica_TC17597 155 156M.esculenta_DV443286 157 158 M.esculenta_TA9427_3983 159 160M.truncatula_CT033771_17.4 161 162 O.minuta_CB884361 163 164O.sativa_LOC_Os01g43230.2 165 166 P.equestris_CB034844 167 168P.glauca_DR564374 169 170 P.hybrida_EB175070 171 172P.menziesii_TA3655_3357 173 174 P.persica_BU039343 175 176P.pinaster_CT579117 177 178 P.pinaster_TA6535_71647 179 180P.sitchensis_TA16538_3332 181 182 P.sitchensis_TA17447_3332 183 184P.taeda_DR096185 185 186 P.tremula_BU888423 187 188 P.tremula_DN497189189 190 P.tremula_TA11725_113636 191 192 P.tremula_TA7610_113636 193 194P.trichocarpa_562293 195 196 P.trichocarpa_568212 197 198P.trichocarpa_594467 199 200 P.trichocarpa_674550 201 202P.trichocarpa_807368 203 204 P.vulgaris_CV538421 205 206S.bicolor_Sb03g028170.1 207 208 S.miltiorrhiza_CV166339 209 210S.miltiorrhiza_TA1626_226208 211 212 S.tuberosum_CV505951 213 214T.aestivum_BE412359 215 216 T.androssowii_TA2313_189785 217 218Triphysaria_sp_TC9313 219 220 V.vinifera_GSVIVT00006915001 221 222V.vinifera_GSVIVT00010755001 223 224 V.vinifera_GSVIVT00026045001 225226 Z.mays_TC409725 227 228

In some instances, related sequences have tentatively been assembled andpublicly disclosed by research institutions, such as The Institute forGenomic Research (TIGR; beginning with TA). The Eukaryotic GeneOrthologs (EGO) database may be used to identify such related sequences,either by keyword search or by using the BLAST algorithm with thenucleic acid sequence or polypeptide sequence of interest. On otherinstances, special nucleic acid sequence databases have been created forparticular organisms, such as by the Joint Genome Institute. Further,access to proprietary databases, has allowed the identification of novelnucleic acid and polypeptide sequences.

1.4. Brassinazole Resistant1 (BZR1)

Sequences (full length cDNA, ESTs or genomic) related BZR nucleic acidsequence were identified amongst those maintained in the EntrezNucleotides database at the National Center for BiotechnologyInformation (NCBI) using database sequence search tools, such as theBasic Local Alignment Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol.215:403-410; and Altschul et al. (1997) Nucleic Acids Res.25:3389-3402). The program was used to find regions of local similaritybetween sequences by comparing nucleic acid or polypeptide sequences tosequence databases and by calculating the statistical significance ofmatches. SEQ ID NO: 2 was used for the TBLASTN algorithm, under defaultsettings and without filters to ignore low complexity sequences. Theoutput of the analysis was viewed by pairwise comparison, and rankedaccording to the probability score (E-value), where the score reflectthe probability that a particular alignment occurs by chance (the lowerthe E-value, the more significant the hit). In addition to E-values,comparisons were also scored by percentage identity. Percentage identityrefers to the number of identical nucleotides (or amino acids) betweenthe two compared nucleic acid (or polypeptide) sequences over aparticular length.

Table A4 provides a list of BZR nucleic acid sequences and encodedproteins thereof.

TABLE A4 Examples of BZR polypeptides: Nucleic Poly- acid peptide SEQSEQ Name Plant Source ID NO: ID NO: AT3G50750.1 Arabidopsis thaliana 238239 AT1G19350.1 Arabidopsis thaliana 240 241 AT1G75080.1 Arabidopsisthaliana 242 243 AT1G78700.1 Arabidopsis thaliana 244 245 AT4G18890.1Arabidopsis thaliana 246 247 AT4G36780.1 Arabidopsis thaliana 248 249Gm\1762729 Glycine max 250 251 Gm\1765606 Glycine max 252 253 Gm\1768381Glycine max 254 255 Gm\1768507 Glycine max 256 257 Hv_TA37786_4513Hordeum vulgare 258 259 Le\LAT61 Lycopersicum esculentum 260 261Le_DB718708 Lycopersicum esculentum 262 263 Le_TA37112_4081 Lycopersicumesculentum 264 265 Le_TA51962_4081 Lycopersicum esculentum 266 267Mt_BF635822 Medicago truncatula 268 269 Mt_TA21345_3880 Medicagotruncatula 270 271 Mt_TA28179_3880 Medicago truncatula 272 273Os01g0203000 Oryza sativa 274 275 Os02g0129600 Oryza sativa 276 277Os06g0552300 Oryza sativa 278 279 Os07g0580500 Oryza sativa 280 281Pp\17189 Physcomitrella patens 282 283 Pp\172161 Physcomitrella patens284 285 Pp\82495 Physcomitrella patens 286 287 Ps\WS0287_023 Piceasitchensis 288 289 Pt\WS01123_K11 Populus trichocarpa 290 291Pt_scaff_178.36 Populus trichocarpa 292 293 Pt_scaff_40.175 Populustrichocarpa 294 295 Pt_scaff_57.215 Populus trichocarpa 296 297Pt_scaff_II.1237 Populus trichocarpa 298 299 Pt_scaff_IV.340 Populustrichocarpa 300 301 Pt_scaff_VII.1038 Populus trichocarpa 302 303Pt_scaff_XI.678 Populus trichocarpa 304 305 Pt_scaff_XI.792 Populustrichocarpa 306 307 Sl\FC26BA11 Solanum lycopersicum 308 309Vv_TA44770_29760 Vitis vinifera 310 311 Zm_AY107201 Zea mays 312 313Zm_EE158804 Zea mays 314 315 Zm_TA175044_4577 Zea mays 316 317Zm_TA178991_4577 Zea mays 318 319

Example 2 Alignment of Sequences Related to the Nucleic Acid SequenceUsed in the Methods of the Invention 2.1. Root Hairless 1 (RNL1)

Alignment of the RHL1 polypeptide sequences of Table A was performedusing the Clustal W algorithm of progressive alignment (Larking et al.Bioinformatics. 2007 Nov. 1; 23(21):2947-8. Thompson et al. (1997)Nucleic Acids Res 25:4876-4882; Chenna et al. (2003). Nucleic Acids Res31:3497-3500). Default values are for the gap open penalty of 10, forthe gap extension penalty of 0.1 and the selected weight matrix isBlosum 62. Proteins alignment is given in FIG. 2 a.

A phylogenetic tree the RHL1 polypeptide sequences of Table A (FIG. 2 b)was constructed using a neighbour-joining clustering algorithm asprovided in the Clustal W programme.

2.2. Transglutaminases (TGases)

Multiple sequence alignment of all the TGase polypeptide sequences inTable A was performed using the AlignX algorithm (from Vector NTI 10.3,Invitrogen Corporation). Results of the alignment are shown in FIG. 7 ofthe present application. The N-terminal plastidic transit peptide isseparated from the rest of the polypeptide by a vertical bar. Theputative calcium binding region is boxed, and marked with X's under theconsensus sequence. The domain where at least one coiled coil ispredicted using the Coils algorithm (and as represented by SEQ ID NO:70) is marked with X's under the consensus region.

2.3. Tryptichon (TRY-Like)

Alignment of polypeptide sequences was performed using the ClustalW 2.0algorithm of progressive alignment (Thompson et al. (1997) Nucleic AcidsRes 25:4876-4882; Chenna et al. (2003). Nucleic Acids Res 31:3497-3500)with standard setting (slow alignment, similarity matrix: Gonnet, gapopening penalty 10, gap extension penalty: 0.2). Minor manual editingwas done to further optimise the alignment. Sequence conservation amongTRY-like polypeptides is essentially in the DNA binding domain of thepolypeptides, the C-terminus and N-terminus usually being more variablein sequence length and composition. The TRY-like polypeptides arealigned in FIG. 10.

This alignment can be used for determining conserved signature sequencesof about 5 to 10 amino acids in length. Preferably the conserved regionsof the proteins are used, recognisable by the asterisks (identicalresidues), the colons (highly conserved substitutions) and the dots(conserved substitutions).

2.4. Brassinazole Resistant1 (BZR1)

Alignment of polypeptide sequences was performed using the AlignXprogramme from the Vector NTI (Invitrogen) which is based on the popularClustal W algorithm of progressive alignment (Thompson et al. (1997)Nucleic Acids Res 25:4876-4882; Chenna et al. (2003). Nucleic Acids Res31:3497-3500). Default values were as follows: gap open penalty of 10;gap extension penalty of 0.1; and the selected weight matrix was Blosum62 (if polypeptides are aligned). The alignment of BZR polypeptides isshown in FIG. 12. The sequence Pp172161 in the alignment is truncated inthe N- and C-terminal. The highly conserved amino acid residues areindicated in the consensus sequence.

Sequence conservation among BZR polypeptides is essentially in theN-terminal part along the BZR1, transcriptional repressor domain. Thehighly conserved bHLH-like DNA binding domain characteristic of BZRpolypeptides is highlighted in FIG. 12. Conserved amino acid motifs suchas SAPVTPPLSSP (SEQ ID NO: 323: located at position 405-415 in theconsensus sequence) and VKPWEGERIHE (SEQ ID NO: 324: located at position634-644 in the consensus sequence) and DLELTLG (SEQ ID NO: 325: locatedat positions 656-662 in the consensus sequence) were identified.

Example 3 Calculation of Global Percentage Identity Between PolypeptideSequences Useful in Performing the Methods of the Invention 3.1. RootHairless 1 (RNL1)

Global percentages of similarity and identity between full lengthpolypeptide sequences useful in performing the methods of the inventionwere determined using one of the methods available in the art, theMatGAT (Matrix Global Alignment Tool) software (BMC Bioinformatics. 20034:29. MatGAT: an application that generates similarity/identity matricesusing protein or DNA sequences. Campanella J J, Bitincka L, Smalley J;software hosted by Ledion Bitincka). MatGAT software generatessimilarity/identity matrices for DNA or protein sequences withoutneeding pre-alignment of the data. The program performs a series ofpair-wise alignments using the Myers and Miller global alignmentalgorithm (with a gap opening penalty of 12, and a gap extension penaltyof 2), calculates similarity and identity using for example Blosum 62(for polypeptides), and then places the results in a distance matrix.Sequence similarity is shown in the bottom half of the dividing line andsequence identity is shown in the top half of the diagonal dividingline.

Parameters used in the comparison were:

-   -   Scoring matrix: Blosum62    -   First Gap: 12    -   Extending gap: 2

Results of the software analysis are shown in Table B1 for the globalsimilarity and identity over the full length of the polypeptidesequences. Percentage identity is given above the diagonal in bold andpercentage similarity is given below the diagonal (normal face).

The percentage identity between the RHL1polypeptide sequences useful inperforming the methods of the invention can be as low as 31% amino acididentity compared to SEQ ID NO: 2.

TABLE B1 MatGAT results for global similarity and identity over the fulllength of the polypeptid sequences. Polypeptide nr Name polypeptide 1 23 4 5 6 7 8 9 10 11 1 O. sativa_Os07g07580 100 88 74 42 42 49 39 40 3935 34 2 O. sativa_Os06g51380 88 100 66 36 35 43 34 35 35 31 30 3 Z.mays_TA180670 74 66 100 38 37 47 38 37 38 33 31 4 A. formosa_TA10038 4236 38 100 60 62 50 50 49 34 35 5 V. vinifera_GSVIVT00027050001 42 35 3760 100 65 51 49 49 27 27 6 M. domestica_TA43921 49 43 47 62 65 100 58 5956 41 39 7 S. tuberosum_TA36268 39 34 38 50 51 58 100 48 50 31 32 8 P.trichocarpa_scaff_IV.277 40 35 37 50 49 59 48 100 49 29 31 9 A.thaliana_AT1G48380.1 39 35 38 49 49 56 50 49 100 32 31 10 P.patens_74926 35 31 33 34 27 41 31 29 32 100 52 11 P. patens_173149 34 3031 35 27 39 32 31 31 52 100

3.2. Transglutaminases (TGases)

Global percentages of similarity and identity between full lengthpolypeptide sequences useful in performing the methods of the inventionwere determined using one of the methods available in the art, theMatGAT (Matrix Global Alignment Tool) software (BMC Bioinformatics. 20034:29. MatGAT: an application that generates similarity/identity matricesusing protein or DNA sequences. Campanella J J, Bitincka L, Smalley J;software hosted by Ledion Bitincka). MatGAT software generatessimilarity/identity matrices for DNA or protein sequences withoutneeding pre-alignment of the data. The program performs a series ofpair-wise alignments using the Myers and Miller global alignmentalgorithm (with a gap opening penalty of 12, and a gap extension penaltyof 2), calculates similarity and identity using for example Blosum 62(for polypeptides), and then places the results in a distance matrix.Sequence similarity is shown in the bottom half of the dividing line andsequence identity is shown in the top half of the diagonal dividingline.

Parameters used in the comparison were:

-   -   Scoring matrix: Blosum62    -   First Gap: 12    -   Extending gap: 2

Results of the software analysis are shown in Table B2 for the globalsimilarity and identity over the full length of the polypeptidesequences (excluding the partial polypeptide sequences).

The percentage identity between the full length polypeptide sequencesuseful in performing the methods of the invention can be as low as 26%amino acid identity compared to SEQ ID NO: 44.

TABLE B MatGAT results for global similarity and identity over the fulllength of the polypeptide sequences of Table A. 1 2 3 4 5 6 7 8 9 10 1112 13  1. Arath_TGase 36 41 26 31 33 30 24 24 21 23 18 16  2.Horvu_TGase 49 40 29 61 30 26 28 27 26 27 22 21  3. Lyces_TGase 58 51 2936 30 26 28 28 27 28 21 21  4. Orysa_TGase 42 46 45 28 26 29 67 62 57 6240 36  5. Orysa_TGase\II 43 69 47 42 25 23 27 27 26 25 20 20  6.Picsi_TGAse 49 41 42 43 34 32 24 22 20 21 17 16  7. Poptr_TGase 50 40 3945 35 51 31 29 26 28 23 22  8. Sacof_TGase 40 45 45 75 42 41 44 88 75 8148 44  9. Sorbi_TGase 40 46 46 70 41 38 41 91 81 87 49 45 10.Zeama_TGase\II 34 43 42 64 42 34 37 77 82 93 49 47 11. Zeama_TGase\III38 46 44 70 41 37 39 83 88 93 50 46 12. Zeama_tgz15 32 37 38 49 37 29 3456 56 58 57 91 13. Zeama_tgz21 29 34 35 45 34 27 32 52 51 55 53 91

The percentage amino acid identity can be significantly increased if themost conserved region of the polypeptides are compared. For example,when comparing the amino acid sequence of the coiled coil domain of SEQID NO: 2 (as represented by SEQ ID NO: 27), with the coiled coil domainof the polypeptides of Table A, the percentage amino acid identityincreased up to 50%.

3.3. Tryptichon (TRY-Like)

Global percentages of similarity and identity between full lengthpolypeptide sequences useful in performing the methods of the inventionwere determined using one of the methods available in the art, theMatGAT (Matrix Global Alignment Tool) software (BMC Bioinformatics. 20034:29. MatGAT: an application that generates similarity/identity matricesusing protein or DNA sequences. Campanella J J, Bitincka L, Smalley J;software hosted by Ledion Bitincka). MatGAT software generatessimilarity/identity matrices for DNA or protein sequences withoutneeding pre-alignment of the data. The program performs a series ofpair-wise alignments using the Myers and Miller global alignmentalgorithm (with a gap opening penalty of 12, and a gap extension penaltyof 2), calculates similarity and identity using for example Blosum 62(for polypeptides), and then places the results in a distance matrix.Sequence similarity is shown in the bottom half of the dividing line andsequence identity is shown in the top half of the diagonal dividingline.

Parameters used in the comparison were:

-   -   Scoring matrix: Blosum62    -   First Gap: 12    -   Extending gap: 2

Results of the software analysis are shown in Table B3 for the globalsimilarity and identity over the full length of the polypeptidesequences. Percentage identity between At5g53200 and other TRY-likepolypeptides is given above the diagonal in bold, for both the fulllength sequence and for the DNA-binding domain.

The percentage identity between the TRY-like polypeptide sequencesuseful in performing the methods of the invention can be as low as 12%sequence identity compared to SEQ ID NO: 76. However, the sequenceconservation is much higher when the DNA binding is compared. Table B2shows similarity and identity among the sequences representing the DNAbinding domain (sequences that align with the DNA-binding domain asshown in FIG. 9 (residues 30 to 75 in SEQ ID NO: 76). The sequenceidentity is generally higher than 50%.

TABLE B3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24A: MatGAT results for global similarity and identity over the fulllength of the polypeptide sequences.  1. Pt_scaff_XII.135 38.1 57.3 19.136.1 15.7 43.3 15.3 13.2 15.1 51.8 52.0 10.3 12.7 31.5 34.0 52.6 12.863.0 56.0 11.8 37.1 12.2 13.8  2. Pt_scaff_64.55 52.6 36.5 15.5 93.212.9 40.7 14.8 12.3 12.8 32.1 41.2 10.6 11.3 31.5 34.9 45.8 11.4 31.138.3 10.4 41.8 11.1 13.3  3. Pt_scaff_II.1572 70.2 44.2 20.1 36.5 16.544.8 16.5 17.5 14.6 47.4 48.1 11.2 13.5 32.3 33.7 45.2 14.7 52.9 51.313.5 33.7 13.7 15.1  4. Pt_scaff_I.1021 29.9 22.2 30.9 16.0 37.8 15.544.3 36.1 40.8 19.1 16.0 29.8 35.3 15.5 15.5 19.6 35.0 21.1 20.1 28.416.0 37.4 38.7  5. Pt_scaff_VII.231 49.5 94.6 43.3 21.1 12.0 38.3 14.812.7 11.9 33.0 39.3 10.6 10.9 32.4 36.1 47.6 11.0 32.1 37.2 10.1 39.211.1 12.8  6. AT3G13540 22.1 17.3 25.7 51.4 17.7 12.4 40.4 35.2 38.216.9 14.1 33.7 45.0 14.5 15.3 13.5 33.9 16.1 14.9 36.7 14.1 36.8 41.0 7. AT4G01060 62.9 62.3 57.7 26.3 63.6 19.7 12.8 14.5 14.6 36.6 45.2 9.711.6 38.3 42.4 65.9 11.0 39.8 48.9 11.5 46.3 11.8 12.8  8. AT5G1475025.1 23.6 30.5 57.1 23.6 53.4 22.2 55.0 58.9 17.2 15.8 28.0 35.3 18.220.2 14.3 31.3 18.1 16.2 31.2 19.2 31.5 38.7  9. AT3G27920 24.1 19.327.2 50.9 21.5 49.0 21.1 64.5 63.8 18.8 14.0 28.8 35.3 16.2 15.8 13.232.5 18.3 15.8 32.8 15.8 31.8 35.9 10. AT5G40330 24.7 19.2 22.4 54.318.3 52.6 21.0 71.2 75.9 17.8 14.5 27.1 34.2 17.1 15.8 13.7 34.3 19.016.0 30.6 16.4 35.6 36.3 11. AT2G30420 65.2 42.9 60.7 32.0 43.8 27.753.6 28.6 28.9 29.2 59.8 13.1 15.6 27.6 31.3 44.6 15.4 57.5 44.6 12.831.0 16.2 15.6 12. AT2G30432 72.2 53.6 60.6 24.2 51.2 20.1 66.7 28.122.4 24.7 66.1 9.0 12.4 28.8 35.3 51.8 11.7 56.5 44.7 10.4 32.6 10.713.8 13. Os03g29614 16.5 13.4 19.0 38.3 13.7 43.9 14.3 41.7 40.5 38.319.6 15.6 37.3 10.2 9.7 9.0 39.6 12.1 11.8 33.7 8.1 40.3 31.2 14.Os01g50110 21.5 16.7 21.8 44.7 16.0 57.1 18.2 46.9 48.7 44.4 21.5 17.848.6 12.7 11.6 12.0 37.4 15.3 14.2 35.1 12.4 37.5 34.5 15. Os01g4318051.4 46.7 57.9 27.8 44.9 21.7 53.3 28.6 26.8 27.4 53.6 53.3 17.1 19.654.5 35.1 11.4 27.4 33.6 13.4 49.5 11.1 15.8 16. Os01g43230 54.6 49.451.9 24.7 50.6 21.7 59.0 27.6 23.7 23.7 48.2 60.7 15.0 16.4 64.5 37.212.8 32.1 37.9 10.8 60.2 12.5 15.9 17. AT1G01380 67.0 59.0 61.5 28.459.0 19.7 79.5 22.7 20.6 21.0 56.3 69.0 14.3 18.2 52.3 59.0 12.7 44.454.3 10.1 39.8 12.1 12.8 18. Zm_C1 19.8 16.1 21.6 44.3 15.4 54.2 17.242.9 45.8 46.9 23.8 19.8 48.6 50.5 20.1 17.9 19.4 13.9 13.9 34.4 10.381.8 37.7 19. At5g53200 79.2 40.6 69.8 33.5 42.5 25.7 55.7 30.5 28.127.9 72.3 64.2 18.4 22.2 55.1 50.9 57.5 20.1 47.7 14.2 30.2 15.9 16.120. AT2G46410 72.2 50.0 66.3 30.4 51.1 23.7 60.6 24.6 25.4 23.7 60.767.0 16.5 21.8 54.2 55.3 66.0 20.1 65.1 12.8 39.4 15.9 16.5 21.Zm_TA175111 18.8 15.6 20.5 44.1 14.9 52.1 17.0 45.8 43.4 44.1 22.6 16.348.3 50.0 19.1 16.3 16.7 45.5 20.8 19.4 9.7 36.1 48.6 22. Zm_TA21830655.7 62.8 50.0 25.3 61.5 19.7 62.8 25.1 23.2 23.7 46.4 64.3 13.1 17.857.0 72.3 59.0 16.1 50.0 57.4 17.4 10.7 13.3 23. Zm_AY135018 20.7 16.619.9 45.0 15.5 53.9 17.7 44.3 48.0 49.1 24.7 18.5 50.8 50.2 19.9 18.818.5 84.6 22.5 20.3 49.7 16.6 36.2 24. Zm_TA175105 25.2 22.0 27.1 55.021.1 57.0 22.0 53.2 52.6 52.5 28.9 22.5 41.7 49.1 25.7 24.8 22.9 47.626.6 25.2 59.0 23.4 46.5 B: MatGAT results for global similarity andidentity over the DNA binding domain of the polypeptide sequences.  1.Zm_C1 95.7 71.7 73.9 91.3 84.8 87.0 89.1 76.1 76.1 73.9 50.0 52.2 52.252.2 47.8 52.2 52.2 47.8 47.8 46.9 47.8 54.3 54.3  2. Zm_AY135018 100.069.6 71.7 91.3 82.6 84.8 91.3 76.1 76.1 73.9 47.8 54.3 54.3 50.0 45.750.0 54.3 50.0 45.7 44.9 45.7 54.3 54.3  3. Zm_TA175111 87.0 89.1 91.367.4 71.7 67.4 71.7 73.9 71.7 71.7 41.3 45.7 47.8 47.8 43.5 47.8 45.743.5 47.8 46.9 45.7 45.7 45.7  4. Zm_TA175105 91.3 91.3 93.5 73.9 73.969.6 73.9 76.1 73.9 73.9 39.1 43.5 45.7 45.7 41.3 45.7 43.5 41.3 45.744.9 43.5 45.7 45.7  5. Os03g29614 95.7 95.7 87.0 93.5 82.6 84.8 89.171.7 71.7 69.6 45.7 50.0 50.0 47.8 43.5 47.8 50.0 45.7 43.5 42.9 43.554.3 54.3  6. AT3G13540 93.5 93.5 87.0 91.3 91.3 87.0 84.8 78.3 78.376.1 50.0 50.0 52.2 54.3 47.8 52.2 50.0 50.0 52.2 51.0 52.2 52.2 52.2 7. Os01g50110 91.3 91.3 84.8 89.1 89.1 91.3 89.1 71.7 73.9 69.6 47.847.8 50.0 52.2 45.7 50.0 47.8 50.0 47.8 46.9 47.8 52.2 52.2  8.Pt_scaff_I.1021 93.5 93.5 93.5 95.7 91.3 93.5 93.5 80.4 82.6 78.3 50.056.5 54.3 52.2 47.8 52.2 54.3 50.0 45.7 44.9 45.7 52.2 52.2  9.AT5G14750 89.1 89.1 93.5 95.7 87.0 89.1 87.0 95.7 97.8 97.8 43.5 50.047.8 52.2 50.0 52.2 45.7 43.5 50.0 46.9 50.0 45.7 45.7 10. AT5G4033089.1 89.1 93.5 95.7 87.0 89.1 87.0 95.7 100.0 95.7 41.3 47.8 45.7 50.047.8 50.0 45.7 43.5 47.8 44.9 47.8 43.5 43.5 11. AT3G27920 89.1 89.193.5 95.7 87.0 89.1 87.0 95.7 100.0 100.0 43.5 50.0 47.8 52.2 50.0 52.245.7 43.5 50.0 46.9 50.0 47.8 47.8 12. Pt_scaff_XII.135 67.4 67.4 63.067.4 65.2 65.2 67.4 71.7 67.4 67.4 67.4 87.0 73.9 78.3 73.9 78.3 67.478.3 65.2 53.1 63.0 65.2 65.2 13. Pt_scaff_II.1572 71.7 71.7 69.6 71.769.6 69.6 71.7 76.1 71.7 71.7 71.7 97.8 78.3 78.3 78.3 82.6 69.6 71.758.7 51.0 54.3 67.4 67.4 14. AT2G46410 71.7 71.7 67.4 69.6 69.6 71.773.9 73.9 69.6 69.6 69.6 89.1 87.0 65.2 63.0 71.7 78.3 73.9 65.2 55.163.0 65.2 65.2 15. AT2G30420 69.6 69.6 65.2 69.6 67.4 67.4 67.4 73.969.6 69.6 69.6 89.1 91.3 87.0 87.0 84.8 60.9 67.4 56.5 53.1 56.5 58.758.7 16. AT2G30432 65.2 65.2 60.9 65.2 63.0 63.0 63.0 69.6 69.6 69.669.6 95.7 93.5 84.8 93.5 89.1 60.9 65.2 54.3 53.1 50.0 56.5 56.5 17.At5g53200 69.6 69.6 65.2 69.6 67.4 67.4 67.4 73.9 69.6 69.6 69.6 97.895.7 91.3 91.3 93.5 63.0 69.6 58.7 53.1 54.3 60.9 60.9 18. AT4G0106073.9 73.9 69.6 71.7 71.7 71.7 71.7 73.9 71.7 69.6 71.7 91.3 89.1 91.391.3 87.0 89.1 78.3 67.4 59.2 67.4 56.5 56.5 19. AT1G01380 69.6 69.663.0 65.2 67.4 67.4 69.6 69.6 65.2 63.0 67.4 91.3 89.1 91.3 84.8 84.887.0 91.3 60.9 51.0 58.7 63.0 63.0 20. Os01g43180 71.7 71.7 69.6 73.969.6 69.6 69.6 73.9 73.9 73.9 73.9 89.1 91.3 87.0 84.8 89.1 91.3 89.184.8 73.5 78.3 58.7 58.7 21. Os01g43230 61.2 61.2 59.2 63.3 59.2 59.259.2 63.3 63.3 63.3 63.3 73.5 73.5 71.4 67.3 71.4 73.5 73.5 69.4 83.765.3 51.0 51.0 22. Zm_TA218306 71.7 71.7 69.6 73.9 69.6 69.6 69.6 73.973.9 73.9 73.9 80.4 80.4 82.6 76.1 76.1 78.3 84.8 76.1 87.0 71.4 58.758.7 23. Pt_scaff_64.55 67.4 67.4 67.4 73.9 67.4 67.4 69.6 67.4 67.467.4 67.4 78.3 76.1 82.6 69.6 69.6 71.7 78.3 80.4 80.4 65.3 82.6 97.824. Pt_scaff_VII.231 69.6 69.6 69.6 76.1 69.6 69.6 71.7 69.6 69.6 69.669.6 78.3 76.1 82.6 69.6 69.6 71.7 78.3 80.4 80.4 65.3 82.6 97.8

3.4. Brassinazole Resistant1 (BZR1)

Global percentages of similarity and identity between full length BZRpolypeptide sequences were determined the MatGAT (Matrix GlobalAlignment Tool) software (BMC Bioinformatics. 2003 4:29. MatGAT: anapplication that generates similarity/identity matrices using protein orDNA sequences. Campanella J J, Bitincka L, Smalley J; software hosted byLedion Bitincka). MatGAT software generates similarity/identity matricesfor DNA or protein sequences without needing pre-alignment of the data.The program performed a series of pair-wise alignments using the Myersand Miller global alignment algorithm (with a gap opening penalty of 12,and a gap extension penalty of 2) and calculated similarity and identityusing for example Blosum 62 (for polypeptides), and then placed theresults in a distance matrix. Sequence similarity is shown in the bottomhalf of the dividing line and sequence identity is shown in the top halfof the diagonal dividing line.

Parameters used in the comparison were:

-   -   Scoring matrix: Blosum62    -   First Gap: 12    -   Extending gap: 2

Results of the software analysis are shown in Table B for the globalsimilarity and identity over the full length of the polypeptidesequences. Percentage identity is given above the diagonal in bold andpercentage similarity is given below the diagonal (normal face).

The percentage identity between the BZR polypeptide sequences of tableB4 compared to SEQ ID NO: 239 ranges from 22.5% to 88.2%.

TABLE B4 MatGAT results for global similarity and identity over the fulllength of the polypeptide sequences. 1 2 3 4 5 6 8 9 10 11 20 21 22 23 1. AT1G19350.1 88.2 40.7 51.9 39.8 25.1 65.4 41.0 54.7 54.9 40.6 23.241.1 56.5  2. AT1G75080.1 92.3 39.4 53.4 39.3 24.0 65.7 40.9 55.4 55.540.6 23.0 40.5 55.1  3. AT1G78700.1 56.1 56.5 46.0 62.1 25.1 40.6 67.146.2 46.7 59.5 22.8 59.3 45.5  4. AT3G50750.1 62.7 64.0 55.1 47.3 33.154.3 42.9 58.6 60.3 40.6 22.5 40.9 57.4  5. AT4G18890.1 54.6 53.3 72.361.6 27.9 42.1 57.2 47.0 48.7 52.2 22.1 52.2 46.2  6. AT4G36780.1 35.235.4 33.2 42.0 36.6 26.5 24.7 29.0 29.8 23.6 18.7 26.5 28.0  8.Gm\1762729 78.5 78.0 55.7 67.2 55.6 37.0 43.1 56.9 56.1 41.9 24.6 41.655.6  9. Gm\1765606 58.8 58.6 80.8 53.6 68.3 31.1 56.6 45.4 44.4 56.622.6 58.4 43.9 10. Gm\1768381 69.9 71.7 59.1 71.3 57.4 36.1 73.3 56.993.6 41.9 21.4 44.0 63.1 11. Gm\1768507 69.9 69.3 56.6 72.1 56.5 37.771.4 56.6 96.1 40.1 21.0 43.2 64.0 20. Os01g0203000 53.4 52.1 69.9 49.962.2 31.2 52.1 69.6 52.6 52.1 23.3 64.8 43.4 21. Os02g0129600 36.9 37.238.2 34.8 35.3 26.2 36.1 37.2 35.9 34.0 37.4 23.3 22.6 22. Os06g055230053.5 52.7 71.0 51.5 62.3 31.8 53.5 70.7 56.3 55.2 78.4 36.6 43.5 23.Os07g0580500 69.6 69.0 58.5 71.1 58.1 37.2 71.1 56.3 75.2 76.3 54.2 35.155.2

Example 4 Identification of Domains Comprised in Polypeptide SequencesUseful in Performing the Methods of the Invention 4.1. Root Hairless 1(RNL1)

Identification of highly conserved sequence motifs in the RNL1polypeptides of Table A was carried out using the MEME system (Timothyet al; 1998. Journal of Computational Biology, Vol. 5, pp. 211-221,1998); Timothy et al. 1998. Bioinformatics, Vol. 14, pp. 48-54).

TABLE C1MEME scan results (major accession numbers) of the polypeptide sequenceas represented by SEQ ID NO: 2. Position of motif in SEQ ID NO: Motif2: Start E-value** Sequence* Motif 1 63 1.5e-118[IV]R[RK][KG][SG]QRK[NS][RK][FY]LFSFPGLLAP Motif 2 85 3.2e-141SGG[KR][IV]G[ED]L[KA]DL[GD]TKNP[ILV]LYLDFPQG [RQ]MKL Motif 3 2446.9e-073 TP[VS]RQSARTAGKK[FL][KN][FY][AT]ExSS Motif 4 155 9.6e-072GTK[ED]ENPEE[LA][RK]L[DE]FPKE[LF]Q[ENQ][GD] Motif 5 34 2.3e-061[SN][GN][NL]L[LQV][SR][EDG]xP[AS][KA]PR[SA][APS]LAPSK[TAG]VL[KR][HL][HQ]G[KR]D Motif 6 177 1.1e-036HA[ED][CY]DFKGGAGAA[CS]D[ES][KA]Q Motif 7 198 2.5e-009[KSN][KEP]P[GEK][EKT][KTE][YT][VT][EG][EPST][ELQ]SP[KE][IT][ED][SLV][ED][DI][DV][LS]S[ED][DE][SD] [NDS][LD]K[DK] Motif 8334 7.3e-004 KG[PA]AAKKQRASP[EM][EA]K[HQ]P[TA]G[KI]K *Amino acids givenbetween brackets indicate any of the possible amino acid at such givenposition. **E-value: Expectation value. The number of differentalignments with scores equivalent to or better than a given Score thatare expected to occur in a database search by chance. The lower the Evalue, the more significant the score.

4.2. Tryptichon (TRY-Like)

The Integrated Resource of Protein Families, Domains and Sites(InterPro) database is an integrated interface for the commonly usedsignature databases for text- and sequence-based searches. The InterProdatabase combines these databases, which use different methodologies andvarying degrees of biological information about well-characterizedproteins to derive protein signatures. Collaborating databases includeSWISS-PROT, PROSITE, TrEMBL, PRINTS, ProDom and Pfam, Smart andTIGRFAMs. Pfam is a large collection of multiple sequence alignments andhidden Markov models covering many common protein domains and families.Pfam is hosted at the Sanger Institute server in the United Kingdom.Interpro is hosted at the European Bioinformatics Institute in theUnited Kingdom.

The results of the InterPro scan of the polypeptide sequence asrepresented by SEQ ID NO: 76 are presented in Table C2.

TABLE C2 InterPro scan results (major accession numbers) of thepolypeptide sequence as represented by SEQ ID NO: 76. Method AccessionDomain start stop E-value HMMPanther PTHR10641: TRIPTYCHON 32 1061.20E−60 SF26 AND CPC HMMPanther PTHR10641 MYB- 32 106 1.20E−60 RELATEDGene3D G3DSA: no 31 79 3.60E−08 1.10.10.60 description HMMPfam PF00249Myb_DNA- 30 75 5.00E−07 binding superfamily SSF46689 Homeodomain- 26 796.90E−07 like HMMSmart SM00717 SANT 29 77 3.10E−06 ProfileScan PS50090MYB_LIKE 34 71 6.307

4.3. Brassinazole Resistant1 (BZR1)

The Integrated Resource of Protein Families, Domains and Sites(InterPro) database is an integrated interface for the commonly usedsignature databases for text- and sequence-based searches. The InterProdatabase combines these databases, which use different methodologies andvarying degrees of biological information about well-characterizedproteins to derive protein signatures. Collaborating databases includeSWISS-PROT, PROSITE, TrEMBL, PRINTS, ProDom and Pfam, Smart andTIGRFAMs. Pfam is a large collection of multiple sequence alignments andhidden Markov models covering many common protein domains and families.Pfam is hosted at the Sanger Institute server in the United Kingdom.Interpro is hosted at the European Bioinformatics Institute in theUnited Kingdom.

The results of the InterPro scan of the polypeptide sequence asrepresented by SEQ ID NO: 239 are presented in Table C3. The Interprofamily corresponding to the BZR polypeptide is IPR008540.

TABLE C3 InterPro scan results (major accession numbers) of thepolypeptide sequence as represented by SEQ ID NO: 239. Amino acidAccession Accession coordinates on Database number name Evalue SEQ IDNO: 239 Pfam PF05687 DUF822 7.199 × E−89 10-157

Example 5 Prediction of Secondary Structure Features of TGase

Coiled coils usually contain a repeated seven amino acid residue patterncalled a heptad repeat. Coiled coils are important to identify forprotein-protein interactions, such as oligomerization, either ofidentical proteins, of proteins of the same family, or of unrelatedproteins. Recently much progress has been made in computationalprediction of coiled coils from sequence data. Many algorithms wellknown to a person skilled in the art are available at the ExPASyProteomics tools. One of them, COILS, is a program that compares asequence to a database of known parallel two-stranded coiled-coils andderives a similarity score. By comparing this score to the distributionof scores in globular and coiled-coil proteins, the program thencalculates the probability that the sequence will adopt a coiled-coilconformation.

The TGase polypeptide as represented by SEQ ID NO: 45, has at least onepredicted coiled coil domain, with a high probability, in all threewindows (14, 21 and 28) examined. In Table D1, the residue coordinates,residues, the three windows and corresponding probability values areshown. In FIG. 6, is the graphical output of the COILS algorithm on thepolypeptide as represented by SEQ ID NO: 45, where at least onepredicted coiled coil is clearly visible, in all three windows (asrepresented by the three lines).

TABLE D1 Numerical output of the COILS algorithm on the polypeptide asrepresented by SEQ ID NO: 45. The residue coordinates (#), residues, thethree windows and corresponding probability values are shown.Probabilities above 0.9 are shown in bold. # Residue Window 14 ProbWindow 21 Prob Window 28 Prob 29 R e 0.001 e 0.082 e 0.946 30 Q f 0.003f 0.082 f 0.946 31 P g 0.003 g 0.082 g 0.946 32 L a 0.708 a 0.991 a1.000 33 D b 0.708 b 0.991 b 1.000 34 R c 0.708 c 0.991 c 1.000 35 A d0.737 d 0.991 d 1.000 36 A e 0.737 e 0.993 e 1.000 37 T f 0.737 f 0.993f 1.000 38 A g 0.813 g 0.999 g 1.000 39 L a 0.980 a 1.000 a 1.000 40 E b0.980 b 1.000 b 1.000 41 I c 0.980 c 1.000 c 1.000 42 L d 0.996 d 1.000d 1.000 43 E e 0.996 e 1.000 e 1.000 44 K f 0.996 f 1.000 f 1.000 45 K g0.996 g 1.000 g 1.000 46 L a 0.996 a 1.000 a 1.000 47 A b 0.996 b 1.000b 1.000 48 E c 0.996 c 1.000 c 1.000 49 Q d 0.996 d 1.000 d 1.000 50 T e0.996 e 1.000 e 1.000 51 A f 0.996 f 1.000 f 1.000 52 E g 0.996 g 1.000g 1.000 53 A a 0.996 a 1.000 a 1.000 54 E b 0.996 b 1.000 b 1.000 55 K c0.996 c 1.000 c 1.000 56 L d 0.996 d 1.000 d 1.000 57 I e 0.971 e 1.000e 1.000 58 R f 0.971 f 1.000 f 1.000 59 E g 0.971 g 1.000 g 1.000 60 N a0.971 a 1.000 a 1.000 61 Q b 0.971 b 1.000 b 1.000 62 R c 0.971 c 1.000c 1.000 63 L d 0.971 d 1.000 d 1.000 64 A e 0.921 e 0.997 e 1.000 65 S f0.848 f 0.989 f 1.000 66 S g 0.231 g 0.917 g 1.000 67 H a 0.099 a 0.537a 0.999 68 V b 0.179 b 0.379 b 0.979 69 V c 0.662 c 0.379 c 0.979 70 L d0.736 d 0.379 d 0.979 71 R e 0.736 e 0.379 e 0.939 72 Q f 0.736 f 0.379f 0.939 73 D g 0.736 g 0.297 g 0.939 74 I a 0.736 a 0.297 a 0.939 75 V b0.736 b 0.297 b 0.939 76 D c 0.736 c 0.297 c 0.939 77 T d 0.736 d 0.297d 0.939 78 E e 0.736 e 0.297 e 0.939 79 K f 0.736 f 0.297 f 0.939 80 E g0.736 g 0.297 g 0.939 81 M a 0.736 a 0.297 a 0.939 82 Q b 0.736 b 0.297b 0.900 83 M c 0.736 c 0.297 c 0.560 84 I d 0.265 d 0.297 d 0.560 85 R e0.265 e 0.297 e 0.560 86 A f 0.151 f 0.297 f 0.407 87 H g 0.047 g 0.297g 0.123 88 L a 0.047 a 0.297 a 0.123 89 G b 0.026 b 0.297 b 0.123 90 D c0.026 c 0.297 c 0.123 91 V d 0.026 d 0.108 d 0.123 92 Q e 0.013 e 0.108e 0.123 93 T b 0.009 b 0.086 b 0.123 94 E c 0.025 c 0.259 c 0.123 95 T d0.025 d 0.259 d 0.123 96 D e 0.025 e 0.259 e 0.123 97 M f 0.051 f 0.259f 0.123 98 H g 0.159 g 0.259 g 0.124 99 M a 0.517 a 0.259 a 0.124 100 Rb 0.556 b 0.259 b 0.124 101 D c 0.707 c 0.259 c 0.124 102 L d 0.707 d0.259 d 0.124 103 M e 0.707 e 0.259 e 0.124 104 E f 0.707 f 0.259 f0.124 105 R g 0.707 g 0.259 g 0.124 106 M a 0.707 a 0.259 a 0.124 107 Rb 0.707 b 0.259 b 0.124 108 L c 0.707 c 0.259 c 0.124 109 M d 0.707 d0.259 d 0.124 110 E e 0.707 e 0.259 e 0.124 111 A f 0.707 f 0.259 f0.124 112 D g 0.707 g 0.259 g 0.124 113 I a 0.707 a 0.259 a 0.124 114 Qb 0.707 b 0.259 b 0.124 115 A c 0.561 c 0.092 c 0.124 116 G d 0.028 d0.057 d 0.124 117 D b 0.040 b 0.424 b 0.842 118 A c 0.042 c 0.452 c0.842 119 V d 0.074 d 0.452 d 0.918 120 K e 0.349 e 0.639 e 0.918 121 Kf 0.349 f 0.639 f 0.918 122 E g 0.349 g 0.639 g 0.918 123 L a 0.349 a0.639 a 0.918 124 H b 0.349 b 0.639 b 0.918 125 Q c 0.349 c 0.639 c0.918 126 V d 0.349 d 0.639 d 0.918 127 H e 0.349 e 0.639 e 0.918 128 Mf 0.349 f 0.639 f 0.918 129 E g 0.349 g 0.797 g 0.918 130 A a 0.349 a0.797 a 0.918 131 K b 0.349 b 0.930 b 0.918 132 R c 0.349 c 0.930 c0.918 133 L d 0.349 d 0.930 d 0.918 134 I e 0.190 e 0.930 e 0.918 135 Af 0.190 f 0.930 f 0.918 136 E g 0.190 g 0.930 g 0.918 137 R a 0.190 a0.930 a 0.918 138 Q b 0.376 b 0.930 b 0.918 139 M c 0.376 c 0.930 c0.918 140 L d 0.426 d 0.930 d 0.918 141 T e 0.426 e 0.930 e 0.918 142 Vf 0.426 f 0.930 f 0.918 143 E g 0.426 g 0.930 g 0.918 144 M a 0.426 a0.930 a 0.918 145 D b 0.426 b 0.930 b 0.918 146 K c 0.426 c 0.930 c0.918 147 V d 0.426 d 0.930 d 0.918 148 T e 0.426 e 0.930 e 0.838 149 Kf 0.426 f 0.930 f 0.838 150 E g 0.426 g 0.930 g 0.838 151 L a 0.426 a0.930 a 0.838 152 H b 0.426 b 0.708 b 0.838 153 K c 0.426 c 0.708 c0.838 154 F d 0.066 d 0.334 d 0.838 155 S e 0.055 e 0.334 e 0.838 156 Gf 0.055 f 0.099 f 0.792 157 D g 0.018 g 0.062 g 0.492 158 S e 0.071 e0.755 e 0.146 159 K f 0.176 f 0.908 f 0.201 160 K g 0.176 g 0.908 g0.288 161 L a 0.333 a 0.908 a 0.288 162 P b 0.333 b 0.908 b 0.288 163 Ec 0.804 c 0.916 c 0.288 164 L d 0.804 d 0.916 d 0.288 165 L e 0.804 e0.916 e 0.288 166 T f 0.807 f 0.916 f 0.288 167 E g 0.856 g 0.916 g0.288 168 L a 0.856 a 0.916 a 0.288 169 D b 0.856 b 0.916 b 0.288 170 Gc 0.856 c 0.916 c 0.288 171 L d 0.856 d 0.916 d 0.288 172 R e 0.856 e0.916 e 0.288 173 K f 0.856 f 0.916 f 0.288 174 E g 0.856 g 0.916 g0.288 175 H a 0.856 a 0.916 a 0.288 176 Q b 0.856 b 0.916 b 0.288 177 Sc 0.856 c 0.916 c 0.288 178 L d 0.856 d 0.916 d 0.288 179 R e 0.856 e0.916 e 0.288 180 S f 0.856 f 0.916 f 0.288 181 A g 0.405 g 0.916 g0.288 182 F a 0.102 a 0.916 a 0.288 183 E b 0.102 b 0.916 b 0.288 184 Yc 0.016 c 0.283 c 0.288 185 E c 0.085 c 0.283 c 0.288 186 K d 0.085 d0.283 d 0.638 187 N e 0.085 e 0.283 e 0.638 188 T f 0.085 f 0.058 f0.638 189 N g 0.085 g 0.025 g 0.638 190 I a 0.085 a 0.026 a 0.638 191 Kb 0.085 b 0.232 b 0.938 192 Q c 0.085 c 0.232 c 0.951 193 V d 0.085 d0.696 d 0.988 194 E f 0.087 f 0.967 f 1.000 195 Q g 0.087 g 0.967 g1.000 196 M a 0.087 a 0.967 a 1.000 197 R b 0.087 b 0.967 b 1.000 198 Tc 0.087 c 0.967 c 1.000 199 M d 0.203 d 0.967 d 1.000 200 E e 0.497 e0.967 e 1.000 201 M f 0.497 f 0.967 f 1.000 202 N g 0.497 g 0.994 g1.000 203 L a 0.497 a 0.994 a 1.000 204 M b 0.497 b 0.994 b 1.000 205 Tc 0.497 c 0.997 c 1.000 206 M d 0.792 d 0.997 d 1.000 207 T e 0.884 e0.997 e 1.000 208 K f 0.993 f 0.997 f 1.000 209 E g 0.993 g 0.997 g1.000 210 A a 0.993 a 0.997 a 1.000 211 D b 0.993 b 0.997 b 1.000 212 Kc 0.993 c 0.997 c 1.000 213 L d 0.993 d 0.997 d 1.000 214 R e 0.993 e0.997 e 1.000 215 A f 0.993 f 0.997 f 1.000 216 D g 0.993 g 0.997 g1.000 217 V a 0.993 a 0.997 a 1.000 218 A b 0.993 b 0.997 b 1.000 219 Nc 0.993 c 0.997 c 1.000 220 A d 0.993 d 0.997 d 1.000 221 E e 0.993 e0.997 e 1.000 222 K f 0.993 f 0.997 f 1.000 223 R g 0.978 g 0.997 g0.999 224 A a 0.978 a 0.997 a 0.999 225 Q b 0.978 b 0.997 b 0.999 226 Vc 0.745 c 0.996 c 0.999 227 A d 0.560 d 0.996 d 0.999 228 A e 0.348 e0.989 e 0.999 229 A f 0.348 f 0.982 f 0.999 230 Q g 0.348 g 0.968 g0.999 231 A a 0.296 a 0.968 a 0.999 232 V g 0.184 g 0.778 g 0.999 233 Aa 0.184 a 0.632 a 0.999 234 A b 0.184 b 0.598 b 0.999 235 Q c 0.184 c0.598 c 0.999 236 A d 0.184 d 0.598 d 0.998 237 G e 0.013 e 0.069 e0.840 238 V f 0.004 f 0.069 f 0.840 239 A b 0.001 b 0.069 b 0.742 240 Hc 0.001 c 0.017 c 0.383 241 V d 0.001 d 0.007 d 0.085 242 T e 0.001 e0.001 e 0.042 243 A f 0.001 f 0.001 f 0.042 244 S g 0.000 g 0.001 g0.013 245 Q f 0.000 f 0.001 f 0.002 246 P b 0.000 b 0.000 b 0.000

Example 6 Topology Prediction of the Polypeptide Sequences Useful inPerforming the Methods of the Invention 6.1. Transglutaminases (TGases)

Many algorithms can be used to perform prediction of the subcellularlocalisation of polypeptides, including:

-   -   TargetP 1.1 hosted on the server of the Technical University of        Denmark;    -   ChloroP 1.1 hosted on the server of the Technical University of        Denmark;    -   Protein Prowler Subcellular Localisation Predictor version 1.2        hosted on the server of the Institute for Molecular Bioscience,        University of Queensland, Brisbane, Australia;    -   PENCE Proteome Analyst PA-GOSUB 2.5 hosted on the server of the        University of Alberta, Edmonton, Alberta, Canada;    -   TMHMM, hosted on the server of the Technical University of        Denmark

By comparing the polypeptide sequence of SEQ ID NO: 45 with orthologsfrom other plant species for which subcellular localisation wasidentified, it is possible to deduce that the subcellular localizationof the polypeptide sequence as represented by SEQ ID NO: 45 is thechloroplast (Villalobos et al. (2004) Gene 336: 93-104).

6.2. Tryptichon (TRY-Like)

TargetP 1.1 predicts the subcellular location of eukaryotic proteins.The location assignment is based on the predicted presence of any of theN-terminal pre-sequences: chloroplast transit peptide (cTP),mitochondrial targeting peptide (mTP) or secretory pathway signalpeptide (SP). Scores on which the final prediction is based are notreally probabilities, and they do not necessarily add to one. However,the location with the highest score is the most likely according toTargetP, and the relationship between the scores (the reliability class)may be an indication of how certain the prediction is. The reliabilityclass (RC) ranges from 1 to 5, where 1 indicates the strongestprediction. TargetP is maintained at the server of the TechnicalUniversity of Denmark.

For the sequences predicted to contain an N-terminal presequence apotential cleavage site can also be predicted.

A number of parameters were selected, such as organism group (non-plantor plant), cutoff sets (none, predefined set of cutoffs, oruser-specified set of cutoffs), and the calculation of prediction ofcleavage sites (yes or no).

The results of TargetP 1.1 analysis of the polypeptide sequence asrepresented by SEQ ID NO: 76 are presented Table E1. The “plant”organism group has been selected, no cutoffs defined, and the predictedlength of the transit peptide requested. The subcellular localization ofthe polypeptide sequence as represented by SEQ ID NO: 76 may be thecytoplasm or nucleus, no transit peptide is predicted.

TABLE E1 TargetP 1.1 analysis of the polypeptide sequence as representedby SEQ ID NO: 76 Length (AA) 106 Chloroplastic transit peptide 0.048Mitochondrial transit peptide 0.348 Secretory pathway signal peptide0.046 Other subcellular targeting 0.822 Predicted Location / Reliabilityclass 3 Predicted transit peptide length /

When analysed with PSort, the probability for a nuclear localisation is0.700, therefore the protein is likely a nuclear protein.

Many other algorithms can be used to perform such analyses, including:

-   -   ChloroP 1.1 hosted on the server of the Technical University of        Denmark;    -   Protein Prowler Subcellular Localisation Predictor version 1.2        hosted on the server of the Institute for Molecular Bioscience,        University of Queensland, Brisbane, Australia;    -   PENCE Proteome Analyst PA-GOSUB 2.5 hosted on the server of the        University of Alberta, Edmonton, Alberta, Canada;    -   TMHMM, hosted on the server of the Technical University of        Denmark    -   PSORT (URL: psort.org)    -   PLOC (Park and Kanehisa, Bioinformatics, 19, 1656-1663, 2003).

Example 7 Assay Related to the Polypeptide Sequences Useful inPerforming the Methods of the Invention 7.1. Functional Assay of RootHairless 1 (RHL1)

The binding of an RHL1 polypeptide is assayed in an in vitro assayessentially as described by Sugimoto-Shirasu et al. 2005 PNAS vol. 102no. 51, 18736-18741. Briefly, Recombinant RHL1 Pprotein is produced in abacterial system and purified using standard methods. Purified RHL1protein is incubated with DNa fragments and binding of the RHL1 proteinthe DNA fragment is detected using plasmon resonance (SPR).

7.2. Transglutaminases (TGases)

Polypeptides useful in performing the methods of the invention typicallycatalyze the formation of amide linkages, generally in a Ca-dependentfashion, between the primary amine of an amine donor substrate and they-carboxamide group of peptide-bound endo-glutamine residues in proteinsor polypeptides that are the amine acceptors. More specifically, TGaseactivity can be measured using the radiolabeled putrescine method, orthe gamma-glutamyl biotin cadaverine method, as described in Villaloboset al. (2004; supra).

A person skilled in the art is well aware of such experimentalprocedures to measure TGase polypeptide enzymatic activity, includingthe activity of a TGase polypeptide as represented by SEQ ID NO: 45.

7.3. Functional Assay of Tryptichon (TRY-Like)

TRY-like polypeptides typically have DNA-binding activity. One methodfor measuring and characterising DNA-binding properties of polypeptidesis described in Xue (A CELD-fusion method for rapid determination of theDNA-binding sequence specificity of novel plant DNA-binding proteins.Plant Journal 41, 638-649, 2005).

Example 8 Cloning of the Nucleic Acid Sequence Used in the Methods ofthe Invention

8.1. Root Hairless 1 (RNL1) The nucleic acid sequence used in themethods of the invention was amplified by PCR using as template acustom-made Arabidopsis thaliana seedlings cDNA library (in pCMV Sport6.0; Invitrogen, Paisley, UK). PCR was performed using Hifi Taq DNApolymerase in standard conditions, using 200 ng of template in a 50 μlPCR mix. The primers used were:5′-ggggacaagtttgtacaaaaaagcaggcttaaacaatggtacgagcttcatcgtc-3′ (SEQ IDNO: 40; sense) and 5′-ggggaccactttgtacaagaaagctgggtttctggaaaagatttctttaagc-3′ (SEQ ID NO: 41; reverse) which include the AttB sites forGateway recombination. The amplified PCR fragment was purified alsousing standard methods. The first step of the Gateway procedure, the BPreaction, was then performed, during which the PCR fragment recombinesin vivo with the pDONR201 plasmid to produce, according to the Gatewayterminology, an “entry clone”, pArath_RHL1. Plasmid pDONR201 waspurchased from Invitrogen, as part of the Gateway® technology.

The entry clone comprising SEQ ID NO: 1 was then used in an LR reactionwith a destination vector used for Oryza sativa transformation. Thisvector contained as functional elements within the T-DNA borders: aplant selectable marker; a screenable marker expression cassette; and aGateway cassette intended for LR in vivo recombination with the nucleicacid sequence of interest already cloned in the entry clone. A rice GOS2promoter (SEQ ID NO: 42) for root specific expression was locatedupstream of this Gateway cassette.

After the LR recombination step, the resulting expression vectorpGOS2::Arath_RHL1l (FIG. 3) was transformed into Agrobacterium strainLBA4044 according to methods well known in the art.

8.2. Transglutaminases (TGases)

The Oryza sativa nucleic acid sequence encoding a TGase polypeptidesequence as represented by SEQ ID NO: 45 was amplified by PCR using astemplate a cDNA bank constructed using RNA from rice plants at differentdevelopmental stages. The following primers, which include the AttBsites for Gateway recombination, were used for PCR amplification:prm02265 (SEQ ID NO: 73, sense): 5′-ggggacaagtttgtacaaaaaagcaggcttcacaatggcataccatggacag-3′ and prm02266 (SEQ ID NO: 74, reverse,complementary): 5′-ggggaccactttgtacaagaaagctgggtatttcacctctggcctg-3′.PCR was performed using Hifi Taq DNA polymerase in standard conditions.A PCR fragment of the expected length (including attB sites) wasamplified and purified also using standard methods. The first step ofthe Gateway procedure, the BP reaction, was then performed, during whichthe PCR fragment recombined in vivo with the pDONR201 plasmid toproduce, according to the Gateway terminology, an “entry clone”. PlasmidpDONR201 was purchased from Invitrogen, as part of the Gateway®technology.

The entry clone comprising SEQ ID NO: 44 was subsequently used in an LRreaction with a destination vector used for Oryza sativa transformation.This vector contained as functional elements within the T-DNA borders: aplant selectable marker; a screenable marker expression cassette; and aGateway cassette intended for LR in vivo recombination with the nucleicacid sequence of interest already cloned in the entry clone. A ricealpha-globulin promoter (SEQ ID NO: 72) for seed-specific expression waslocated upstream of this Gateway cassette.

After the LR recombination step, the resulting expression vectorpGlob::TGase (FIG. 4) for seed-specific expression, was transformed intoAgrobacterium strain LBA4044 according to methods well known in the art.

8.3. Tryptichon (TRY-Like)

The nucleic acid sequence used in the methods of the invention wasamplified by PCR using as template a custom-made Arabidopsis thalianaseedlings cDNA library (in pCMV Sport 6.0; Invitrogen, Paisley, UK). PCRwas performed using Hifi Taq DNA polymerase in standard conditions,using 200 ng of template in a 50 μl PCR mix. The primers used wereprm09014 (SEQ ID NO: 233; sense, start codon in bold):5′-ggggacaagtttgtacaaaaaagcagg cttaaacaatggataacactgaccgtcgt-3′ andprm09015 (SEQ ID NO: 234; reverse, complementary):5′-ggggaccactttgtacaagaaagctgggttttttcgttggcttaaaaa ca-3′, which includethe AttB sites for Gateway recombination. The amplified PCR fragment waspurified also using standard methods. The first step of the Gatewayprocedure, the BP reaction, was then performed, during which the PCRfragment recombines in vivo with the pDONR201 plasmid to produce,according to the Gateway terminology, an “entry clone”, pTRY-like.Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway®technology.

The entry clone comprising SEQ ID NO: 75 was then used in an LR reactionwith a destination vector used for Oryza sativa transformation. Thisvector contained as functional elements within the T-DNA borders: aplant selectable marker; a screenable marker expression cassette; and aGateway cassette intended for LR in vivo recombination with the nucleicacid sequence of interest already cloned in the entry clone. A rice GOS2promoter (SEQ ID NO: 237) for constitutive specific expression waslocated upstream of this Gateway cassette. In an alternative embodiment,a root specific promoter (RCc3 promoter; SEQ ID NO: 235)

After the LR recombination step, the resulting expression vectorpGOS2::TRY-like (FIG. 3) or pRCc3::TRY was transformed intoAgrobacterium strain LBA4044 according to methods well known in the art.

8.4. Brassinazole Resistant1 (BZR1)

The nucleic acid sequence used in the methods of the invention wasamplified by PCR using as template a custom-made Arabidopsis thalianaseedlings cDNA library (in pCMV Sport 6.0; Invitrogen, Paisley, UK). PCRwas performed using Hifi Taq DNA polymerase in standard conditions,using 200 ng of template in a 50 μl PCR mix. The primers used were (SEQID NO: 320; sense):5′-ggggacaagtttgtacaaaaaagcaggcttaaacaatgacggcatcaggag ga-3′ and (SEQ IDNO: 321; reverse, complementary): 5′-ggggaccactttgtacaagaaagctgggtaccacgatattaacctagccg-3′, which include the AttB sites forGateway recombination. The amplified PCR fragment was purified alsousing standard methods. The first step of the Gateway procedure, the BPreaction, was then performed, during which the PCR fragment recombinedin vivo with the pDONR201 plasmid to produce, according to the Gatewayterminology, an “entry clone”, pBZR. Plasmid pDONR201 was purchased fromInvitrogen, as part of the Gateway® technology.

The entry clone comprising SEQ ID NO: 238 was then used in an LRreaction with a destination vector used for Oryza sativa transformation.This vector contained as functional elements within the T-DNA borders: aplant selectable marker; a screenable marker expression cassette; and aGateway cassette intended for LR in vivo recombination with the nucleicacid sequence of interest already cloned in the entry clone. A rice GOS2promoter (SEQ ID NO: 322) for constitutive specific expression waslocated upstream of this Gateway cassette.

After the LR recombination step, the resulting expression vectorpGOS2::BZR (FIG. 3) was transformed into Agrobacterium strain LBA4044according to methods well known in the art.

Example 9 Plant Transformation Rice Transformation

The Agrobacterium containing the expression vector was used to transformOryza sativa plants. Mature dry seeds of the rice japonica cultivarNipponbare were dehusked. Sterilization was carried out by incubatingfor one minute in 70% ethanol, followed by 30 minutes in 0.2% HgCl₂,followed by a 6 times 15 minutes wash with sterile distilled water. Thesterile seeds were then germinated on a medium containing 2,4-D (callusinduction medium). After incubation in the dark for four weeks,embryogenic, scutellum-derived calli were excised and propagated on thesame medium. After two weeks, the calli were multiplied or propagated bysubculture on the same medium for another 2 weeks. Embryogenic calluspieces were sub-cultured on fresh medium 3 days before co-cultivation(to boost cell division activity).

Agrobacterium strain LBA4404 containing the expression vector was usedfor co-cultivation. Agrobacterium was inoculated on AB medium with theappropriate antibiotics and cultured for 3 days at 28° C. The bacteriawere then collected and suspended in liquid co-cultivation medium to adensity (OD₆₀₀) of about 1. The suspension was then transferred to aPetri dish and the calli immersed in the suspension for 15 minutes. Thecallus tissues were then blotted dry on a filter paper and transferredto solidified, co-cultivation medium and incubated for 3 days in thedark at 25° C. Co-cultivated calli were grown on 2,4-D-containing mediumfor 4 weeks in the dark at 28° C. in the presence of a selection agent.During this period, rapidly growing resistant callus islands developed.After transfer of this material to a regeneration medium and incubationin the light, the embryogenic potential was released and shootsdeveloped in the next four to five weeks. Shoots were excised from thecalli and incubated for 2 to 3 weeks on an auxin-containing medium fromwhich they were transferred to soil. Hardened shoots were grown underhigh humidity and short days in a greenhouse.

Approximately 35 independent T0 rice transformants were generated forone construct. The primary transformants were transferred from a tissueculture chamber to a greenhouse. After a quantitative PCR analysis toverify copy number of the T-DNA insert, only single copy transgenicplants that exhibit tolerance to the selection agent were kept forharvest of T1 seed. Seeds were then harvested three to five months aftertransplanting. The method yielded single locus transformants at a rateof over 50% (Aldemita and Hodges 1996, Chan et al. 1993, Hiei et al.1994).

Corn Transformation

Transformation of maize (Zea mays) is performed with a modification ofthe method described by Ishida et al. (1996) Nature Biotech 14(6):745-50. Transformation is genotype-dependent in corn and only specificgenotypes are amenable to transformation and regeneration. The inbredline A188 (University of Minnesota) or hybrids with A188 as a parent aregood sources of donor material for transformation, but other genotypescan be used successfully as well. Ears are harvested from corn plantapproximately 11 days after pollination (DAP) when the length of theimmature embryo is about 1 to 1.2 mm. Immature embryos are cocultivatedwith Agrobacterium tumefaciens containing the expression vector, andtransgenic plants are recovered through organogenesis. Excised embryosare grown on callus induction medium, then maize regeneration medium,containing the selection agent (for example imidazolinone but variousselection markers can be used). The Petri plates are incubated in thelight at 25° C. for 2-3 weeks, or until shoots develop. The green shootsare transferred from each embryo to maize rooting medium and incubatedat 25° C. for 2-3 weeks, until roots develop. The rooted shoots aretransplanted to soil in the greenhouse. T1 seeds are produced fromplants that exhibit tolerance to the selection agent and that contain asingle copy of the T-DNA insert.

Wheat Transformation

Transformation of wheat is performed with the method described by Ishidaet al. (1996) Nature Biotech 14(6): 745-50. The cultivar Bobwhite(available from CIMMYT, Mexico) is commonly used in transformation.Immature embryos are co-cultivated with Agrobacterium tumefacienscontaining the expression vector, and transgenic plants are recoveredthrough organogenesis. After incubation with Agrobacterium, the embryosare grown in vitro on callus induction medium, then regeneration medium,containing the selection agent (for example imidazolinone but variousselection markers can be used). The Petri plates are incubated in thelight at 25° C. for 2-3 weeks, or until shoots develop. The green shootsare transferred from each embryo to rooting medium and incubated at 25°C. for 2-3 weeks, until roots develop. The rooted shoots aretransplanted to soil in the greenhouse. T1 seeds are produced fromplants that exhibit tolerance to the selection agent and that contain asingle copy of the T-DNA insert.

Soybean Transformation

Soybean is transformed according to a modification of the methoddescribed in the Texas A&M patent U.S. Pat. No. 5,164,310. Severalcommercial soybean varieties are amenable to transformation by thismethod. The cultivar Jack (available from the Illinois Seed foundation)is commonly used for transformation. Soybean seeds are sterilised for invitro sowing. The hypocotyl, the radicle and one cotyledon are excisedfrom seven-day old young seedlings. The epicotyl and the remainingcotyledon are further grown to develop axillary nodes. These axillarynodes are excised and incubated with Agrobacterium tumefacienscontaining the expression vector. After the cocultivation treatment, theexplants are washed and transferred to selection media. Regeneratedshoots are excised and placed on a shoot elongation medium. Shoots nolonger than 1 cm are placed on rooting medium until roots develop. Therooted shoots are transplanted to soil in the greenhouse. T1 seeds areproduced from plants that exhibit tolerance to the selection agent andthat contain a single copy of the T-DNA insert.

Rapeseed/Canola Transformation

Cotyledonary petioles and hypocotyls of 5-6 day old young seedling areused as explants for tissue culture and transformed according to Babicet al. (1998, Plant Cell Rep 17: 183-188). The commercial cultivarWestar (Agriculture Canada) is the standard variety used fortransformation, but other varieties can also be used. Canola seeds aresurface-sterilized for in vitro sowing. The cotyledon petiole explantswith the cotyledon attached are excised from the in vitro seedlings, andinoculated with Agrobacterium (containing the expression vector) bydipping the cut end of the petiole explant into the bacterialsuspension. The explants are then cultured for 2 days on MSBAP-3 mediumcontaining 3 mg/I BAP, 3% sucrose, 0.7% Phytagar at 23° C., 16 hr light.After two days of co-cultivation with Agrobacterium, the petioleexplants are transferred to MSBAP-3 medium containing 3 mg/I BAP,cefotaxime, carbenicillin, or timentin (300 mg/I) for 7 days, and thencultured on MSBAP-3 medium with cefotaxime, carbenicillin, or timentinand selection agent until shoot regeneration. When the shoots are 5-10mm in length, they are cut and transferred to shoot elongation medium(MSBAP-0.5, containing 0.5 mg/I BAP). Shoots of about 2 cm in length aretransferred to the rooting medium (MS0) for root induction. The rootedshoots are transplanted to soil in the greenhouse. T1 seeds are producedfrom plants that exhibit tolerance to the selection agent and thatcontain a single copy of the T-DNA insert.

Alfalfa Transformation

A regenerating clone of alfalfa (Medicago sativa) is transformed usingthe method of (McKersie et al., 1999 Plant Physiol 119: 839-847).Regeneration and transformation of alfalfa is genotype dependent andtherefore a regenerating plant is required. Methods to obtainregenerating plants have been described. For example, these can beselected from the cultivar Rangelander (Agriculture Canada) or any othercommercial alfalfa variety as described by Brown D C W and A Atanassov(1985. Plant Cell Tissue Organ Culture 4: 111-112). Alternatively, theRA3 variety (University of Wisconsin) has been selected for use intissue culture (Walker et al., 1978 Am J Bot 65:654-659). Petioleexplants are cocultivated with an overnight culture of Agrobacteriumtumefaciens C58C1 pMP90 (McKersie et al., 1999 Plant Physiol 119:839-847) or LBA4404 containing the expression vector. The explants arecocultivated for 3 d in the dark on SH induction medium containing 288mg/L Pro, 53 mg/L thioproline, 4.35 g/L K2SO4, and 100 μmacetosyringinone. The explants are washed in half-strengthMurashige-Skoog medium (Murashige and Skoog, 1962) and plated on thesame SH induction medium without acetosyringinone but with a suitableselection agent and suitable antibiotic to inhibit Agrobacterium growth.After several weeks, somatic embryos are transferred to BOi2Ydevelopment medium containing no growth regulators, no antibiotics, and50 g/L sucrose. Somatic embryos are subsequently germinated onhalf-strength Murashige-Skoog medium. Rooted seedlings were transplantedinto pots and grown in a greenhouse. T1 seeds are produced from plantsthat exhibit tolerance to the selection agent and that contain a singlecopy of the T-DNA insert.

Cotton Transformation

Cotton is transformed using Agrobacterium tumefaciens according to themethod described in U.S. Pat. No. 5,159,135. Cotton seeds are surfacesterilised in 3% sodium hypochlorite solution during 20 minutes andwashed in distilled water with 500 μg/ml cefotaxime. The seeds are thentransferred to SH-medium with 50 μg/ml benomyl for germination.Hypocotyls of 4 to 6 days old seedlings are removed, cut into 0.5 cmpieces and are placed on 0.8% agar. An Agrobacterium suspension (approx.108 cells per ml, diluted from an overnight culture transformed with thegene of interest and suitable selection markers) is used for inoculationof the hypocotyl explants. After 3 days at room temperature andlighting, the tissues are transferred to a solid medium (1.6 g/IGelrite) with Murashige and Skoog salts with B5 vitamins (Gamborg etal., Exp. Cell Res. 50:151-158 (1968)), 0.1 mg/I 2,4-D, 0.1 mg/I6-furfurylaminopurine and 750 μg/ml MgCL2, and with 50 to 100 μg/mlcefotaxime and 400-500 μg/ml carbenicillin to kill residual bacteria.Individual cell lines are isolated after two to three months (withsubcultures every four to six weeks) and are further cultivated onselective medium for tissue amplification (30° C., 16 hr photoperiod).Transformed tissues are subsequently further cultivated on non-selectivemedium during 2 to 3 months to give rise to somatic embryos. Healthylooking embryos of at least 4 mm length are transferred to tubes with SHmedium in fine vermiculite, supplemented with 0.1 mg/I indole aceticacid, 6 furfurylaminopurine and gibberellic acid. The embryos arecultivated at 30° C. with a photoperiod of 16 hrs, and plantlets at the2 to 3 leaf stage are transferred to pots with vermiculite andnutrients. The plants are hardened and subsequently moved to thegreenhouse for further cultivation.

Example 10 Phenotypic Evaluation Procedure 10.1 Evaluation Setup

Approximately 35 independent T0 rice transformants were generated. Theprimary transformants were transferred from a tissue culture chamber toa greenhouse for growing and harvest of T1 seed. Six events, of whichthe T1 progeny segregated 3:1 for presence/absence of the transgene,were retained. For each of these events, approximately 10 T1 seedlingscontaining the transgene (hetero- and homo-zygotes) and approximately 10T1 seedlings lacking the transgene (nullizygotes) were selected bymonitoring visual marker expression. The transgenic plants and thecorresponding nullizygotes were grown side-by-side at random positions.Greenhouse conditions were of shorts days (12 hours light), 28° C. inthe light and 22° C. in the dark, and a relative humidity of 70%. Plantsgrown under non-stress conditions are watered at regular intervals toensure that water and nutrients are not limiting to satisfy plant needsto complete growth and development.

Four T1 events were further evaluated in the T2 generation following thesame evaluation procedure as for the T1 generation but with moreindividuals per event. From the stage of sowing until the stage ofmaturity the plants were passed several times through a digital imagingcabinet. At each time point digital images (2048×1536 pixels, 16 millioncolours) were taken of each plant from at least 6 different angles.

Drought Screen

Plants from T2 seeds are grown in potting soil under normal conditionsuntil they approached the heading stage. They were then transferred to a“dry” section where irrigation was withheld. Humidity probes wereinserted in randomly chosen pots to monitor the soil water content(SWC). When SWC went below certain thresholds, the plants wereautomatically re-watered continuously until a normal level was reachedagain. The plants were then re-transferred again to normal conditions.The rest of the cultivation (plant maturation, seed harvest) was thesame as for plants not grown under abiotic stress conditions. Growth andyield parameters are recorded as detailed for growth under normalconditions.

Nitrogen Use Efficiency Screen

Rice plants from T2 seeds were grown in potting soil under normalconditions except for the nutrient solution. The pots were watered fromtransplantation to maturation with a specific nutrient solutioncontaining reduced N nitrogen (N) content, usually between 7 to 8 timesless. The rest of the cultivation (plant maturation, seed harvest) wasthe same as for plants not grown under abiotic stress. Growth and yieldparameters are recorded as detailed for growth under normal conditions.

Salt Stress Screen

Plants are grown on a substrate made of coco fibers and argex (3 to 1ratio). A normal nutrient solution was used during the first two weeksafter transplanting the plantlets in the greenhouse. After the first twoweeks, 25 mM of salt (NaCl) was added to the nutrient solution, untilthe plants were harvested. Seed-related parameters were then measured.

10.2 Statistical Analysis: F Test

A two factor ANOVA (analysis of variants) was used as a statisticalmodel for the overall evaluation of plant phenotypic characteristics. AnF test was carried out on all the parameters measured of all the plantsof all the events transformed with the gene of the present invention.The F test was carried out to check for an effect of the gene over allthe transformation events and to verify for an overall effect of thegene, also known as a global gene effect. The threshold for significancefor a true global gene effect was set at a 5% probability level for theF test. A significant F test value points to a gene effect, meaning thatit is not only the mere presence or position of the gene that is causingthe differences in phenotype.

Because two experiments with overlapping events were carried out, acombined analysis was performed. This is useful to check consistency ofthe effects over the two experiments, and if this is the case, toaccumulate evidence from both experiments in order to increaseconfidence in the conclusion. The method used was a mixed-model approachthat takes into account the multilevel structure of the data (i.e.experiment-event-segregants). P values were obtained by comparinglikelihood ratio test to chi square distributions.

10.3 Parameters Measured Biomass-Related Parameter Measurement

From the stage of sowing until the stage of maturity the plants werepassed several times through a digital imaging cabinet. At each timepoint digital images (2048×1536 pixels, 16 million colours) were takenof each plant from at least 6 different angles.

The plant aboveground area (or leafy biomass) was determined by countingthe total number of pixels on the digital images from aboveground plantparts discriminated from the background. This value was averaged for thepictures taken on the same time point from the different angles and wasconverted to a physical surface value expressed in square mm bycalibration. Experiments show that the aboveground plant area measuredthis way correlates with the biomass of plant parts above ground. Theabove ground area is the area measured at the time point at which theplant had reached its maximal leafy biomass. The early vigour is theplant (seedling) aboveground area three weeks post-germination. Increasein root biomass is expressed as an increase in total root biomass(measured as maximum biomass of roots observed during the lifespan of aplant); or as an increase in the root/shoot index (measured as the ratiobetween root mass and shoot mass in the period of active growth of rootand shoot).

Early vigour was determined by counting the total number of pixels fromaboveground plant parts discriminated from the background. This valuewas averaged for the pictures taken on the same time point fromdifferent angles and was converted to a physical surface value expressedin square mm by calibration. The results described below are for plantsthree weeks post-germination.

Seed-Related Parameter Measurements

The mature primary panicles were harvested, counted, bagged,barcode-labelled and then dried for three days in an oven at 37° C. Thepanicles were then threshed and all the seeds were collected andcounted. The filled husks were separated from the empty ones using anair-blowing device. The empty husks were discarded and the remainingfraction was counted again. The filled husks were weighed on ananalytical balance. The number of filled seeds was determined bycounting the number of filled husks that remained after the separationstep. The total seed yield was measured by weighing all filled husksharvested from a plant. Total seed number per plant was measured bycounting the number of husks harvested from a plant. Thousand KernelWeight (TKW) is extrapolated from the number of filled seeds counted andtheir total weight. The Harvest Index (HI) in the present invention isdefined as the ratio between the total seed yield and the above groundarea (mm²), multiplied by a factor 10⁶. The total number of flowers perpanicle as defined in the present invention is the ratio between thetotal number of seeds and the number of mature primary panicles. Theseed fill rate as defined in the present invention is the proportion(expressed as a %) of the number of filled seeds over the total numberof seeds (or florets).

Example 11 Results of the Phenotypic Evaluation of the Transgenic Plants11. 1. Root Hairless 1 (RNL1)

The results of the evaluation of transgenic rice plants in T2 generationexpressing coding region of an Arath_RHL1 nucleic acid (SEQ ID NO: 1)under the growth conditions of nitrogen limitation of Example 8 arepresented below. An increase of at least 5% was observed for emergencevigour (early vigour, EmerVigor), total seed yield (totalweightseeds),number of filled seeds (Nr filled seeds), harvest index (harvestindex),root biomass (Rootmax) and the number of total seeds on a plant(nrtotalseed) (Table F1).

TABLE F1 Evaluation of transgenic plants expressing the Arath_RHL1 geneunder nitrogen limitation growth conditions. % increase in transgenicParameter compared to control plant EmerVigor 19 RootMax 7.6totalweightseeds 17 Nr filled seeds 16 harvestindex 12 nrtotalseed 11

The results of the evaluation of transgenic rice plants in T1 generationexpressing the coding region of Orysa_RHL1 nucleic acid (SEQ ID NO: 9)from the constitutive promoter GOS2 (SEQ ID NO: 39) under the non-stresspresented below (Table F2).

TABLE F2 Evaluation of transgenic plants expressing an Orysa_RHL1nucleic acid under non-stress conditions. % increase in transgenicParameter compared to control plant AreaMax 8.1 TimetoFlower 1.25RootMax 3.6 totalwgseeds 16.19 nrfilledseed 14.9 filtrate 5.7harvestindex 10.0 HeightMax 2.6 GNbfFlow 7.4 nrtotalseed 8.6

The results of the evaluation of transgenic rice plants in T1 generationexpressing the coding region of Orysa_RHL1 nucleic acid (SEQ ID NO: 9)driven from the root specific promoter Rcc3 (SEQ ID NO: 40) grown undernitrogen limiting conditions as specified above in the Nitrogen useefficiency screen are shown in Table F3. EmerVigor (also refer to asEarly vigour) is a yield trait directly correlated with the vigour ofthe plant in particular at early, seedling stage of development.

TABLE F3 Evaluation of transgenic plants expressing an Orysa_RHL1nucleic acid under nitrogen limiting conditions. % increase intransgenic Parameter compared to control plant EmerVigor 16.6totalwgseeds 12 nrfilledseed 15

11.2. Transglutaminases (TGases)

The results of the evaluation of T1 and T2 generation transgenic riceplants expressing the nucleic acid sequence encoding a TGase polypeptideas represented by SEQ ID NO: 45, under the control of a seed-specificpromoter, and grown under normal growth conditions, are presented below.

There was a significant increase in the early vigor, in the abovegroundbiomass, in the total seed yield per plant, in the total number ofseeds, in the number of filled seeds, in the seed filling rate, and inthe harvest index of the transgenic plants compared to correspondingnullizygotes (controls), as shown in Table F4.

TABLE F4 Results of the evaluation of T1 and T2 generation transgenicrice plants expressing the nucleic acid sequence encoding a TGasepolypeptide as represented by SEQ ID NO: 45, under the control of apromoter for seed-specific expression. Overall average % Overall average% increase in 8 events increase in 4 events Trait in the T1 generationin the T2 generation Total seed yield per plant 26% 15% Total number offilled seeds 27% 14% Harvest index 26% 14%

11.3. Tryptichon (TRY-Like)

The evaluation of transgenic rice plants expressing a TRY-like nucleicacid under control of the RCc3 promoter, and grown under conditions ofreduced nitrogen availability, revealed an increase of more than 5% foremergence vigour (early vigour), fill rate, harvest index, and totalseed yield. These increases were observed in T1 generation plants aswell as in T2 generation plants.

The results of the evaluation of transgenic rice plants, expressing anucleic acid encoding the TRY polypeptide of SEQ ID NO: 76 under controlof the constitutive promoter, and grown under non-stress conditions inthe T1 and the T2 generation, are presented below in Table E and Frespectively. When grown under non-stress conditions, an increase of atleast 5% was observed in T1 for seed yield (total weight of seeds,number of filled seeds, number of total seeds).

TABLE F5 Data summary for transgenic rice plants transformed with thepGOS2::TRY construct; for each parameter, the overall percent increaseis shown for the T1 generation, for each parameter the p-value is ≦0.05.Parameter Overall increase Nr total seeds 8.7 totalwgseeds 17.3nrfilledseed 14.5

In the T2 generation, a strong increase 5%) was found for above groundbiomass (AreaMax and firstpan), early vigour, and seed yield; detailsare given in Table F:

TABLE F6 Data summary for transgenic rice plants transformed with thepGOS2::TRY construct; for each parameter, the overall percent increaseis shown for the T2 generation, for each parameter the p-value is ≦0.05.Parameter Overall increase AreaMax 5.0 EmerVigor 20.2 firstpan 8.3totalwgseeds 8.8 nrfilledseed 7.5

11.4. Brassinazole Resistant1 (BZR1)

The results of the evaluation of transgenic rice plants expressing thecoding region of the BZR nucleic acid of SEQ ID NO: 1 under non-stressconditions are presented below. An increase of at least 5% was observedfor total seed yield, the number of filled seeds per plant, the seedfilling rate and the harvest index, and of more than 2.5% for thousandkernel weight compared to control (corresponding nullyzogotes) plants(Table F7).

TABLE F7 Total Seed Number of Seed Harvest Parameter weight filled seedfilling rate Index TKW % increase in the 17.5 14.4 5.5 11.4 3.0transgenic compared to the control plant

1. A method for enhancing yield-related traits in a plant relative to acontrol plant, comprising: (a) modulating expression in a plant of anucleic acid encoding a Root Hairless 1 (RHL1) polypeptide, atransglutaminase (TGase) polypeptide, a TRY-like (tryptichon)polypeptide, or a Brassinazole-Resistant (BZR) polypeptide; and (b)optionally selecting for a plant having enhanced yield-related traitsrelative to a control plant, wherein: (i) said RHL1 polypeptidecomprises one or more of Motif 9 of SEQ ID NO: 37, Motif 10 of SEQ IDNO: 38, and Motif 11 of SEQ ID NO: 39; (ii) said TGase polypeptidecomprises a plastidic transit peptide and has at least 50% sequenceidentity to the amino acid sequence of SEQ ID NO: 27; (iii) saidTRY-like polypeptide comprises a Myb-like DNA-binding domain and/or oneor more of motifs of SEQ ID NO: 229 to SEQ ID NO: 232; or (iv) said BZRpolypeptide comprises a domain having at least 50% sequence identity tothe domain located between amino acid coordinates 10-157 in SEQ ID NO:239, a domain having at least 50% sequence identity to a bHLH-likedomain comprising the amino acid sequence of SEQ ID NO: 326, and/or amotif comprising the amino acid sequence of SEQ ID NO: 323, 324, or 325.2. The method of claim 1, wherein said modulated expression is effectedby introducing and expressing in the plant said nucleic acid.
 3. Themethod of claim 1, wherein: (a) said nucleic acid encoding a RHL1polypeptide encodes any one of the proteins listed in Table A1, or is aportion of such a nucleic acid, or a nucleic acid capable of hybridizingwith such a nucleic acid or the complement thereof; (b) said nucleicacid encoding a TGase polypeptide encodes the TGase polypeptide of SEQID NO: 45 or any one of the proteins listed in Table A2, or is a portionof such a nucleic acid, or a nucleic acid capable of hybridizing withsuch a nucleic acid or the complement thereof; (c) said nucleic acidencoding a TRY-like polypeptide encodes any one of the proteins listedin Table A3, or is a portion of such a nucleic acid, or a nucleic acidcapable of hybridizing with such a nucleic acid; or (d) said nucleicacid encoding a BZR polypeptide encodes any one of the proteins listedin Table A4, or is a portion of such a nucleic acid, or a nucleic acidcapable of hybridizing with such a nucleic acid.
 4. The method of claim1, wherein: (a) said nucleic acid encodes a RHL1 polypeptide, andwherein said enhanced yield-related traits comprise increased seed yieldrelative to a control plant; (b) said nucleic acid encodes a TGasepolypeptide, and wherein said enhanced yield-related traits compriseincreased total seed yield per plant, increased number of filled seeds,and/or increased harvest index relative to a control plant; (c) saidnucleic acid encodes a TRY-like polypeptide, and wherein said enhancedyield-related traits comprise increased emergence vigour and/orincreased yield relative to a control plant; or (d) said nucleic acidencodes a BZR polypeptide, and wherein said enhanced yield-relatedtraits comprise increased total weight of the seed, increased number offilled seed and increased thousand kernel weight relative to a controlplant.
 5. The method of claim 1, wherein: (a) said nucleic acid encodesa RHL1 polypeptide, and wherein said enhanced yield-related traits areobtained under cultivation conditions of nitrogen deficiency; (b) saidnucleic acid encodes a TRY-like polypeptide, and wherein said enhancedyield-related traits are obtained under non-stress conditions or underconditions of nitrogen deficiency; or (c) said nucleic acid encodes aBZR polypeptide, and wherein said enhanced yield-related traits areobtained under non-stress conditions.
 6. The method of claim 1, wherein:(a) said nucleic acid encoding a RHL1 polypeptide is operably linked toa constitutive promoter, a GOS2 promoter, or a GOS2 promoter from rice;(b) said nucleic acid encoding a TGase polypeptide is operably linked toa seed-specific promoter; (c) said nucleic acid encoding a TGasepolypeptide is operably linked to an alpha-globulin promoter, a ricealpha-globulin promoter, or an alpha-globulin promoter comprising thenucleotide sequence of SEQ ID NO: 72; (d) said nucleic acid encoding aTRY-like polypeptide is operably linked to a root-specific promoter, aRCc3 promoter, or a RCc3 promoter from rice; (e) said nucleic acidencoding a TRY-like polypeptide is operably linked to a constitutivepromoter, a GOS2 promoter, or a GOS2 promoter from rice; or (f) saidnucleic acid encoding a BZR polypeptide is operably linked to aconstitutive promoter, a GOS2 promoter, or a GOS2 promoter from rice. 7.The method of claim 1, wherein: (a) said nucleic acid encoding a RHL1polypeptide is of plant origin, from a dicotyledonous plant, from aplant of the family Brassicaceae, or from an Arabidopsis thaliana plant;(b) said nucleic acid encoding a TGase polypeptide is from a plant, froma monocotyledonous plant, from a plant of the family Poaceae, or from anOryza sativa plant; (c) said nucleic acid encoding a TRY-likepolypeptide is of plant origin, from a dicotyledonous plant, from aplant of the family Brassicaceae, from a plant of the genus Arabidopsis,or from an Arabidopsis thaliana plant; or (d) said nucleic acid encodinga BZR polypeptide is of plant origin, from a dicotyledonous plant, froma plant of the family Brasicaceae, or from an Arabidopsis thalianaplant.
 8. A plant obtained by the method of claim 1, or a plant part,seed, or progeny of said plant, wherein said plant, or said plant part,seed, or progeny, comprises a recombinant nucleic acid encoding saidRHL1 polypeptide, said TGase polypeptide, said TRY-like polypeptide, orsaid BZR polypeptide.
 9. Harvestable parts of the plant of claim 8,wherein said harvestable parts comprises said recombinant nucleic acidand are preferably shoot biomass and/or seeds.
 10. Products derived fromthe plant of claim 8 and/or from harvestable parts of said plant,wherein said products comprises said recombinant nucleic acid.
 11. Themethod of claim 1, wherein the plant is a crop plant, a monocot or acereal, or wherein said plant is rice, maize, wheat, barley, millet,rye, triticale, sorghum or oats.
 12. The method of claim 1, wherein saidnucleic acid encodes a BZR polypeptide and comprises: (a) the nucleotidesequence of SEQ ID NO: 238; (b) a nucleotide sequence encoding apolypeptide comprising the amino acid sequence of SEQ ID NO: 239; or (c)a nucleotide sequence encoding a polypeptide having at least 80%sequence identity to the amino acid sequence of SEQ ID NO:
 239. 13. Aconstruct comprising: (a) a nucleic acid sequence encoding a RHL1polypeptide, a TGase polypeptide, a TRY-like polypeptide, or a BZRpolypeptide as defined in claim 1; (b) one or more control sequencescapable of driving expression of the nucleic acid sequence of (a); andoptionally (c) a transcription termination sequence.
 14. The constructof claim 13, wherein: (a) said nucleic acid encodes a RHL1 polypeptide,and wherein said one or more control sequences comprise a constitutivepromoter, a GOS2 promoter, or a GOS2 promoter from rice; (b) saidnucleic acid encodes a TGase polypeptide, and wherein said one or morecontrol sequences comprise a seed-specific promoter; (c) said nucleicacid encodes a TGase polypeptide, and wherein said one or more controlsequences comprise an alpha-globulin promoter, a rice alpha-globulinpromoter, or an alpha-globulin promoter comprising the nucleotidesequence of SEQ ID NO: 72; (d) said nucleic acid encodes a TRY-likepolypeptide, and wherein said one or more control sequences comprise aroot-specific promoter, a RCc3 promoter, or a RCc3 promoter from rice;(e) said nucleic acid encodes a TRY-like polypeptide, and wherein saidone or more control sequences comprise a constitutive promoter, a GOS2promoter, or a GOS2 promoter from rice; or (f) said nucleic acid encodesa BZR polypeptide, and wherein said one or more control sequencescomprise a constitutive promoter, a GOS2 promoter, or a GOS2 promoterfrom rice.
 15. A plant, plant part or plant cell comprising theconstruct of claim
 13. 16. A method for the production of a transgenicplant having increased yield relative to a control plant, comprising:(a) introducing and expressing in a plant or plant cell a nucleic acidencoding a RHL1 polypeptide, a TGase polypeptide, a TRY-likepolypeptide, or a BZR polypeptide as defined in claim 1; (b) cultivatingthe plant or plant cell under conditions promoting plant growth anddevelopment; and optionally (c) selecting for a plant having increasedyield relative to a control plant.
 17. A transgenic plant havingincreased yield relative to a control plant, resulting from modulatedexpression of a nucleic acid encoding a RNL1 polypeptide, a TGasepolypeptide, a TRY-like polypeptide, or a BZR polypeptide as defined inclaim 1, or a transgenic plant cell derived from said transgenic plant.18. The transgenic plant of claim 17, wherein said plant is a cropplant, a monocot or a cereal, or wherein said plant is rice, maize,wheat, barley, millet, rye, triticale, sorghum or oats.
 19. Harvestableparts of the transgenic plant of claim 17, wherein said harvestableparts are preferably shoot biomass and/or seeds.
 20. Products derivedfrom the transgenic plant of claim 17 and/or from harvestable parts ofsaid plant.